G Proteins, Polynucleotides Encoding the Same and Utilization Thereof

ABSTRACT

A novel protein (Gm1) includes an amino acid sequence part having a high homology with a domain having a high homology with a GTP binding site and a GTPase site conserved among G protein α subunits and a trimer forming domain conserved among G protein α subunits. The Gm1 protein is involved in signal transduction via a G protein-coupled receptor (GPCR) stimulation. The Gm1 protein is expressed intensively in human brain, thymus, testes, spleen, small intestine, uterus and heart. A method for screening for a substance capable of regulating a cellular signal transduction employs a polynucleotide encoding the Gm1 protein.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a division of U.S. patent application Ser. No.10/618,320, filed Jul. 11, 2003, the disclosure of which is herebyincorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a novel G protein having an ability ofamplifying a signal transduction generated by a receptor upon binding toa natural ligand such as amine, peptide, hormone, autacoid,neurotransmitter and the like, as well as a polynucleotide encoding thesame. The invention also relates to a method for screening for asubstance which regulates the cellular signal transduction mediated bythis novel G protein, and the like.

2. Description of the Background Art

A G protein is an important mediator in a signal transduction. Thus, theG protein serves as a transmitter which transport, into a cell, astimulation signal received by a G protein-coupled receptor (hereinaftersometimes abbreviated as GPCR) which is a seven transmembrane receptor.A GPCR is expressed in a wide variety of tissues, and this signaltransduction system was proven to be involved in regulation of a widevariety of cellular functions such as hormone reception,neurotransmission, cell proliferation and differentiation and the like(GENDAI KAGAKU ZOKAN 34, 61 to 70, 1997).

More specifically, a G protein is a heterotrimer formed from α subunitwhich binds to GTP, β subunit and γ subunit. When a GPCR on a cellmembrane surface binds to a ligand such as a hormone orneurotransmitter, the GPCR is activated and its signal is transmitted toa G protein. In the G protein to which the signal has been transmitted,a GDP is released from the inactive form in which a GDP was bound onceto the α subunit, and a GTP is then bound instead, whereby convertinginto an active form.

The active G protein is released from the GPCR while dissociating into aGTP-binding α subunit and βγ subunits. The active G protein promotes orinhibits its target effector such as adenylate cyclase, Ca²⁺ channel, K⁺channel, phospholipase Cβ and the like, whereby regulating a variety ofthe cellular functions. A mammalian G protein α subunit may for examplebe Gi, Go, Gq, Gt and the like. Typically, a G protein α subunit isclassified into any of 4 types, namely, the type which promotes theactivity of an adenylate cyclase, the type which inhibits the activityof an adenylate cyclase, the type which promotes the activity of aphospholipase and the type which transmits a signal to a Rho.

A GTP which has been bound to an α subunit of an active G protein isconverted into a GDP by the GTPase effect possessed by the α subunit,resulting in the recovery of an inactive form (“Signal Dentatsu”, p.17-30, Nov. 1, 2001, KYORITSU SHUPPANSHA).

GPCR genes and its gene products and GPCR signal transductionpathway-related genes and its gene products are potential causes fordiseases (Spiegel et al., J. Clin. Invest. 92: 1119-1125 (1993);MuKusick et al., J. Med. Genet. 30:1-26 (1993); Lania et al., EuropeanJ. Endocrinology 145:543-559 (2001)). For example, a certain defect in aV2 vasopressin receptor gene as a GPCR has been proven to induce variousforms of a nephrogenic diabetes insipidus (Holtzman et al., Hum. Mol.Genet. 2:1201-1204 (1993)). In addition, variation in Gα subunits areobserved in a tumor of growth hormone secreting cells in a pituitarygland which secrets a growth hormone, hyperthyroid tumor, ovarian andrenal tumors (Meij, JTA (1996), Mol. Cell. Biochem. 157:31-38; Aussel,C. et al., (1988), J. Immunol. 140: 215-220). Accordingly, GPCRsignal-related gene products are useful as a target of a novel drug, and50% of currently marketed pharmaceuticals were reported to direct theGPCR as a target (Nature Review Drug Discovery, 1, 7 (2002)).

Generally in screening natural ligands of GPCR, it is important to knowwhich G proteins are to be coupled with the GPCR (Trends inPharmacological Science, 22, 560-564 (2001)). Accordingly,identification of a novel G protein and a gene encoding the same isuseful in treating or diagnosing a disease caused by the abnormality inthe cellular signal transduction in which said G protein is involved. Inaddition, it can be used in the screening for a pharmaceutical which isuseful as a remedial, therapeutic or prophylactic agent against adisease caused by the abnormality in a cellular signal transduction.Furthermore, it can be used in the screening for a pharmaceutical whichis useful as a remedial, therapeutic or prophylactic agent capable ofameliorating or preventing a symptom by means of the activation orinhibition of the cellular signal transduction.

SUMMARY OF THE INVENTION

A main object of the present invention is to provide a novel G protein αsubunit and a polynucleotide encoding the same, a substance capable ofactivating or inhibiting the signal transduction system mediated by a Gprotein-coupled receptor and this G protein α subunit, a method forscreening for such a substance as well as a screening kit therefor.

For the purpose of accomplishing the objective described above, we madean effort and finally discovered a human novel protein comprising anamino acid sequence having a high homology with the GTP binding site andthe GTPase activation site conserved among G protein α subunits and anamino acid sequence having a high homology with the heterotrimer formingdomain conserved among the G protein α subunits, and designated thisprotein as a Gm1 protein. We also discovered a mouse Gm1 protein and arat Gm1 protein having similar characteristics with regard to the aminoacid sequences.

Moreover, we discovered that in a cell having a Gm1 expression vector,the effector activity of the G protein is elevated.

Furthermore, we discovered that Gm1 protein is expressed at a high levelin human brain, thymus, testes, spleen, small intestine, uterus andheart.

Based on the findings described above, we believed that the presentprotein (Gm1 protein) is a novel G protein α subunit which is a moleculeinvolved in the signal transduction mediated by a GPCR stimulation whichfunctions in human brain, thymus, testes, spleen, small intestine,uterus and heart, thus establishing the present invention.

Thus, the invention provides the proteins and polynucleotides and thelike, which are listed in the following respective paragraphs:

1. A protein comprising any amino acid sequence selected from the groupconsisting of:

(a) the amino acid sequence represented by SEQ ID NO:1;(b) an amino acid sequence of a protein involved in a G protein-coupledreceptor mediated signal transduction, said protein consists of an aminoacid sequence having a homology of 85% or more with the amino acidsequence represented by SEQ ID NO:1;(c) the amino acid sequence represented by SEQ ID NO:25;(d) the amino acid sequence represented by SEQ ID NO:26;(e) an amino acid sequence of a protein involved in a G protein-coupledreceptor mediated signal transduction, said protein comprises the aminoacid sequence of the amino acid Nos. 96 to 126 of SEQ ID NO:1;(f) an amino acid sequence of a protein involved in a G protein-coupledreceptor mediated signal transduction, said protein comprises an aminoacid sequence having a homology of 95% or more with the amino acidsequence of the amino acid Nos. 96 to 126 of SEQ ID NO:1;(g) an amino acid sequence of a protein involved in a G protein-coupledreceptor mediated signal transduction, said protein comprises at itsN-terminal the amino acid sequence of the amino acid Nos. 1 to 126 ofSEQ ID NO:1; and(h) an amino acid sequence of a protein involved in a G protein-coupledreceptor mediated signal transduction, said protein comprises at itsN-terminal an amino acid sequence having a homology of 65% or more withthe amino acid sequence of the amino acid Nos. 1 to 126 of SEQ ID NO:1.

2. A protein (1) or (2):

(1) the protein consisting of the amino acid sequence represented by SEQID NO:1;(2) a protein involved in a G protein-coupled receptor mediated signaltransduction which consists of an amino acid sequence having a homologyof 85% or more with the amino acid sequence represented by SEQ ID NO:1.

3. The protein consisting of the amino acid sequence represented by SEQID NO:25.

4. The protein consisting of the amino acid sequence represented by SEQID NO:26.

5. A protein (3) or (4):

(3) a protein involved in a G protein-coupled receptor mediated signaltransduction which comprises the amino acid sequence of the amino acidNos. 96 to 126 of SEQ ID NO:1;(4) a protein involved in a G protein-coupled receptor mediated signaltransduction which comprises an amino acid sequence having a homology of95% or more with the amino acid sequence of the amino acid Nos. 96 to126 of SEQ ID NO:1.

6. A protein (5) or (6):

(5) a protein involved in a G protein-coupled receptor mediated signaltransduction which comprises at its N-terminal the amino acid sequenceof the amino acid Nos. 1 to 126 of SEQ ID NO:1;(6) a protein involved in a G protein-coupled receptor mediated signaltransduction which comprises at its N-terminal an amino acid sequencehaving a homology of 65% or more with the amino acid sequence of theamino acid Nos. 1 to 126 of SEQ ID NO:1.

7. A polynucleotide comprising a nucleotide sequence selected from thegroup consisting of:

(a) a nucleotide sequence encoding the amino acid sequence representedby SEQ ID NO:1;(b) a nucleotide sequence encoding an amino acid sequence of a proteininvolved in a G protein-coupled receptor mediated signal transduction,said protein consists of an amino acid sequence having a homology of 85%or more with the amino acid sequence represented by SEQ ID NO:1;(c) a nucleotide sequence encoding the amino acid sequence representedby SEQ ID NO:25;(d) a nucleotide sequence encoding the amino acid sequence representedby SEQ ID NO:26;(e) a nucleotide sequence encoding an amino acid sequence of a proteininvolved in a G protein-coupled receptor mediated signal transduction,said protein comprises the amino acid sequence of the amino acid Nos. 96to 126 of SEQ ID NO:1;(f) a nucleotide sequence encoding an amino acid sequence of a proteininvolved in a G protein-coupled receptor mediated signal transduction,said protein comprises an amino acid sequence having a homology of 95%or more with the amino acid sequence of the amino acid Nos. 96 to 126 ofSEQ ID NO:1;(g) a nucleotide sequence encoding an amino acid sequence of a proteininvolved in a G protein-coupled receptor mediated signal transduction,said protein comprises at its N-terminal the amino acid sequence of theamino acid Nos. 1 to 126 of SEQ ID NO:1;(h) a nucleotide sequence encoding an amino acid sequence of a proteininvolved in a G protein-coupled receptor mediated signal transduction,said protein comprises at its N-terminal an amino acid sequence having ahomology of 65% or more with the amino acid sequence of the amino acidNos. 1 to 126 of SEQ ID NO:1;(i) the nucleotide sequence represented by SEQ ID NO:2;(j) a nucleotide sequence of a polynucleotide encoding a proteininvolved in a G protein-coupled receptor mediated signal transduction,said polynucleotide consists of a nucleotide sequence having a homologyof 85% or more with the polynucleotide consisting of the nucleotidesequence represented by SEQ ID NO:2;(k) the nucleotide sequence represented by SEQ ID NO:27;(l) the nucleotide sequence represented by SEQ ID NO:28;(m) a nucleotide sequence of a polynucleotide encoding a proteininvolved in a G protein-coupled receptor mediated signal transduction,said polynucleotide comprises the nucleotide sequence of the nucleotideNos. 289 to 378 of SEQ ID NO:2;(n) a nucleotide sequence of a polynucleotide encoding a proteininvolved in a G protein-coupled receptor mediated signal transduction,said polynucleotide comprises a nucleotide sequence having a homology of90% or more with the polynucleotide consisting of the nucleotidesequence of the nucleotide Nos. 289 to 378 of SEQ ID NO:2;(o) a nucleotide sequence of a polynucleotide encoding a proteininvolved in a G protein-coupled receptor mediated signal transduction,said polynucleotide comprises at its 5′ terminal the nucleotide sequenceof the nucleotide Nos. 1 to 378 of SEQ ID NO:2; and(p) a nucleotide sequence of a polynucleotide encoding a proteininvolved in a G protein-coupled receptor mediated signal transduction,said polynucleotide comprises at its 5′ terminal a nucleotide sequencehaving a homology of 70% or more with the polynucleotide consisting ofthe nucleotide sequence of the nucleotide Nos. 1 to 378 of SEQ ID NO:2.

8. A polynucleotide of (7) or (8):

(7) the polynucleotide consisting of the nucleotide sequence representedby SEQ ID NO:2;(8) a polynucleotide encoding a protein involved in a G protein-coupledreceptor mediated signal transduction, said polynucleotide consists of anucleotide sequence having a homology of 85% or more with thepolynucleotide consisting of the nucleotide sequence represented by SEQID NO:2.

9. The polynucleotide consisting of the nucleotide sequence representedby SEQ ID NO:27.

10. The polynucleotide consisting of the nucleotide sequence representedby SEQ ID NO:28.

11. A polynucleotide of (9) or (10):

(9) a polynucleotide encoding a protein involved in a G protein-coupledreceptor mediated signal transduction, said polynucleotide comprises thenucleotide sequence of the nucleotide Nos. 289 to 378 of SEQ ID NO:2;(10) a polynucleotide encoding a protein involved in a G protein-coupledreceptor mediated signal transduction, said polynucleotide comprises anucleotide sequence having a homology of 90% or more with thepolynucleotide consisting of the nucleotide sequence of the nucleotideNos. 289 to 378 of SEQ ID NO:2.

12. A polynucleotide of (11) or (12):

(11) a polynucleotide encoding a protein involved in a G protein-coupledreceptor mediated signal transduction, said polynucleotide comprises atits 5′ terminal the nucleotide sequence of the nucleotide Nos. 1 to 378of SEQ ID NO:2;(12) a polynucleotide encoding a protein involved in a G protein-coupledreceptor mediated signal transduction, said polynucleotide comprises atits 5′ terminal a nucleotide sequence having a homology of 70% or morewith the polynucleotide consisting of the nucleotide sequence of thenucleotide Nos. 1 to 378 of SEQ ID NO:2.

13. A recombinant vector containing a polynucleotide according to theabove 7.

14. A method for producing a recombinant vector comprising a step forintegrating a polynucleotide according to the above 7 into a vectorcapable of being replicated in a host cell.

15. A transformant having a recombinant vector according to the above13.

16. A method for producing a transformant comprising a step fortransducing a recombinant vector according to the above 13 into a hostcell.

17. A method for producing a G protein α-subunit comprising steps forculturing a transformant having a recombinant vector containing apolynucleotide according to the above 7 and recovering from the culturea protein resulting from the polynucleotide according to the above 7.

18. An antisense polynucleotide consisting of a polynucleotide of (13)or (14):

(13) a polynucleotide which inhibits the expression of a proteinaccording to the above 1 which comprises a nucleotide sequencecomplementary to at least 15 contiguous nucleotides in the nucleotidesequence represented by SEQ ID NO:2;(14) a polynucleotide which inhibits the expression of a proteinaccording to the above 1 which hybridizes under an intracellularcondition with a polynucleotide consisting of at least 15 contiguousnucleotides in the nucleotide sequence represented by SEQ ID NO:2.

19. A ribozyme (15) or (16):

(15) a ribozyme having an ability of cleaving a polynucleotide accordingto the above 7 which comprises two polynucleotide regions complementaryto two regions respectively consisting of at least 9 contiguousnucleotides which are two regions in the nucleotide sequence representedby SEQ ID NO:2;(16) a ribozyme having an ability of cleaving a polynucleotide accordingto the above 7 which comprises two polynucleotide regions whichhybridizes under an intracellular condition with two regionsrespectively consisting of at least 9 contiguous nucleotides which aretwo regions in the nucleotide sequence represented by SEQ ID NO:2.

20. An antibody which recognizes a protein according to the above 1specifically.

21. An agent for regulating a G protein-coupled receptor mediated signaltransduction containing as an active ingredient a protein according tothe above 1.

22. A therapeutic or prophylactic agent against a disease caused by a Gprotein-coupled receptor mediated signal transduction abnormality,wherein an active ingredient of the agent is a protein according to theabove 1.

23. An agent for regulating a G protein-coupled receptor mediated signaltransduction containing as an active ingredient a polynucleotideaccording to the above 7.

24. A therapeutic or prophylactic agent against a disease caused by a Gprotein-coupled receptor mediated signal transduction abnormality,wherein an active ingredient of the agent is a polynucleotide accordingto the above 7.

25. An agent for regulating a G protein-coupled receptor mediated signaltransduction containing as an active ingredient an antisensepolynucleotide according to the above 18.

26. A therapeutic or prophylactic agent against a disease caused by a Gprotein-coupled receptor mediated signal transduction abnormality,wherein an active ingredient of the agent is an antisense polynucleotideaccording to the above 18.

27. An agent for regulating a G protein-coupled receptor mediated signaltransduction containing as an active ingredient a ribozyme according tothe above 19.

28. A therapeutic or prophylactic agent against a disease caused by a Gprotein-coupled receptor mediated signal transduction abnormality,wherein an active ingredient of the agent is a ribozyme according to theabove 19.

29. An agent for regulating a G protein-coupled receptor mediated signaltransduction containing as an active ingredient an antibody according tothe above 20.

30. A therapeutic or prophylactic agent against a disease caused by a Gprotein-coupled receptor mediated signal transduction abnormality,wherein an active ingredient of the agent is an antibody according tothe above 20.

31. An oligonucleotide (17) or (18):

(17) an oligonucleotide capable of recognizing a polynucleotiderepresented by SEQ ID NO:2 specifically which consists of at least 17contiguous nucleotides in the nucleotide sequence represented by SEQ IDNO:2;(18) an oligonucleotide capable of recognizing a polynucleotiderepresented by SEQ ID NO:2 specifically which has a homology of 80% ormore with at least 17 contiguous nucleotides in the nucleotide sequencerepresented by SEQ ID NO:2.

32. An oligonucleotide according to the above 31 which is used as aprobe or a primer.

33. A method for screening for a substance capable of regulating asignal transduction mediated by a G protein-coupled receptor and a Gprotein comprising:

(a) a step for bringing a test substance into contact with a test cellhaving a recombinant vector according to the above 13 and a recombinantvector containing a DNA encoding a G protein-coupled receptor protein;(b) a step for measuring the G protein effector activity or the indexvalue correlating therewith in the test cell; and(c) a step for comparing this effector activity or the index valuecorrelating therewith the effector activity or the index valuecorrelating therewith in the test cell which has not been brought intocontact with the test substance, whereby selecting a test substancecapable of altering the effector activity or the index value correlatingtherewith in the test cell.

34. A method for screening for a substance capable of regulating asignal transduction mediated by a G protein-coupled receptor and a Gprotein comprising:

(a) a step for bringing a test substance into contact with a test cellhaving a recombinant vector according to the above 13 and a recombinantvector containing a DNA encoding a G protein-coupled receptor protein;(b) a step for measuring the G protein effector activity or the indexvalue correlating therewith in the test cell; and(c) a step for comparing this effector activity with the effectoractivity or the index value correlating therewith when the said testsubstance has been brought into contact with a control cell having norecombinant vector according to the above 13 but having a recombinantvector containing a DNA encoding a G protein-coupled receptor protein,whereby selecting a test substance causing a difference in the effectoractivity or the index value correlating therewith between the test celland the control cell.

35. A method for screening for a substance capable of regulating asignal transduction mediated by a G protein-coupled receptor and a Gprotein comprising:

(a) a step for bringing a test substance into contact with a test cellhaving a recombinant vector according to the above 13 and a recombinantvector containing a DNA encoding a G protein-coupled receptor protein;(b) a step for measuring the G protein effector activity or the indexvalue correlating therewith in the test cell; and(c) a step for comparing this effector activity or the index valuecorrelating therewith the effector activity or the index valuecorrelating therewith when the said test substance has been brought intocontact with a control cell having no recombinant vector containing aDNA encoding a G protein-coupled receptor protein but having arecombinant vector according to the above 13, whereby selecting a testsubstance causing a difference in the effector activity or the indexvalue correlating therewith between the test cell and the control cell.

36. A method for screening for a substance capable of regulating asignal transduction mediated by a G protein-coupled receptor and a Gprotein comprising:

(a) a step for bringing a test substance and a G protein-coupledreceptor ligand into contact with a test cell having a recombinantvector according to the above 13 and a recombinant vector containing aDNA encoding a G protein-coupled receptor protein;(b) a step for measuring the G protein effector activity or the indexvalue correlating therewith in the test cell; and(c) a step for comparing this effector activity or the index valuecorrelating therewith the effector activity or the index valuecorrelating therewith in the test cell which has not been brought intocontact with the test substance but has been brought into contact withthe ligand, whereby selecting a test substance capable of altering theeffector activity or the index value correlating therewith in the testcell.

37. A method for screening for a substance capable of regulating asignal transduction mediated by a G protein-coupled receptor and a Gprotein comprising:

(a) a step for bringing a test substance and a G protein-coupledreceptor ligand into contact with a test cell having a recombinantvector according to the above 13 and a recombinant vector containing aDNA encoding a G protein-coupled receptor protein;(b) a step for measuring the G protein effector activity or the indexvalue correlating therewith in the test cell;(c) a step for comparing this effector activity with the effectoractivity in the test cell which has not been brought into contact withthe test substance but has been brought into contact with the ligand,whereby investigating the change in the effector activity in the testcell; and(d) a step for comparing the rate of change in this effector activity orthe index value correlating therewith the rate of change in the effectoractivity or the index value correlating therewith when the said testsubstance and said ligand has been brought into contact with a controlcell having no recombinant vector containing a DNA encoding a Gprotein-coupled receptor protein but having a recombinant vectoraccording to the above 13, whereby selecting a test substance causing adifference in the rate of change in the effector activity or the indexvalue correlating therewith between the test cell and the control cell.

38. A method for screening for a substance capable of regulating asignal transduction mediated by a G protein-coupled receptor and a Gprotein comprising:

(a) a step for bringing a test substance into contact with a cellmembrane fraction of a cell having a recombinant vector according to theabove 13 and a cell membrane fraction of a cell having a recombinantvector containing a DNA encoding a GPCR, ora cell membrane fraction of a cell having the recombinant vectoraccording to the above 13 and the recombinant vector containing the DNAencoding the GPCR;(b) a step for assaying the level of the binding of GTP to the cellmembrane fraction; and(c) a step for comparing the assayed level of this GTP binding with theassayed level of the GTP binding to the cell membrane fraction which hasnot been brought into contact with the test substance, whereby selectinga test substance capable of altering the assayed level of the GTPbinding to the cell membrane fraction.

39. A method for screening for a substance capable of regulating asignal transduction mediated by a G protein-coupled receptor and a Gprotein comprising:

(a) a step for bringing a test substance and a G protein-coupledreceptor ligand into contact witha cell membrane fraction of a cell having a recombinant vector accordingto the above 13 and a cell membrane fraction of a cell having arecombinant vector containing a DNA encoding a GPCR, ora cell membrane fraction of a cell having the recombinant vectoraccording to the above 13 and the recombinant vector containing the DNAencoding the GPCR;(b) a step for assaying the level of the binding of GTP to the cellmembrane fraction; and(c) a step for comparing the assayed level of this GTP binding with theassayed level of the GTP binding in the cell membrane fraction which hasnot been brought into contact with the test substance but has beenbrought into contact with said ligand, whereby selecting a testsubstance capable of altering the assayed level of the GTP binding tothe cell membrane fraction.

40. A method for screening for a substance capable of regulating asignal transduction mediated by a G protein-coupled receptor and a Gprotein comprising:

(a) a step for bringing a test substance into contact with a test cellcapable of expressing a protein according to the above 1;(b) a step for measuring the expression level of the protein accordingto the above 1 in the test cell; and(c) a step for comparing this expression level with the expression levelof said protein in the test cell which has not been brought into contactwith the test substance, whereby selecting a test substance capable ofaltering the expression level of said protein in the test cell.

41. A substance capable of regulating a signal transduction mediated bya G protein-coupled receptor and a G protein, said substance is obtainedby a screening method according to any of the above 33 to 40.

42. An agent for regulating a signal transduction mediated by a Gprotein-coupled receptor and a G protein, said agent contains as anactive ingredient a substance according to the above 41.

43. A therapeutic or prophylactic agent against a disease caused by theabnormality in a G protein-coupled receptor and a G protein-mediatedsignal transduction containing as an active ingredient a substanceaccording to the above 41.

44. A kit for screening for a substance capable of regulating a signaltransduction mediated by a G protein-coupled receptor and a proteinaccording to the above 1, which comprises a test cell having arecombinant vector containing a polynucleotide encoding a proteinaccording to the above 1 and a reagent for measuring the G proteineffector activity or an index value correlating therewith.

45. A screening kit according to the above 44 wherein the test cellfurther has a recombinant vector having a DNA encoding a Gprotein-coupled receptor.

46. A screening kit according to the above 44 further containing a Gprotein-coupled receptor ligand.

47. A screening kit according to the above 44 further containing acontrol cell having a recombinant vector having a DNA encoding a Gprotein-coupled receptor.

48. A screening kit according to the above 44 further containing acontrol cell having a recombinant vector containing a polynucleotideencoding a protein according to the above 1.

49. A kit for screening for a substance capable of regulating a signaltransduction mediated by a G protein-coupled receptor and a proteinaccording to the above 1, which comprises:

a cell having a recombinant vector containing a polynucleotide encodinga protein according to the above 1; and,

a GTP analogue which can bind to the protein according to the above 1but can not be cleaved by a GTPase.

50. A screening kit according to the above 49 wherein the cell furtherhas a recombinant vector having a DNA encoding a G protein-coupledreceptor.

51. A kit for screening for a substance capable of regulating a signaltransduction mediated by a G protein-coupled receptor and a proteinaccording to the above 1, which comprises:

a cell having a recombinant vector containing a polynucleotide encodinga protein according to the above 1;

a cell having a recombinant vector having a DNA encoding the Gprotein-coupled receptor; and,

a GTP analogue which can bind to the protein according to the above 1but can not be cleaved by a GTPase.

52. A screening kit according to the above 49 or 51 further containing aG protein-coupled receptor ligand.

53. A kit for screening for a substance capable of regulating a signaltransduction mediated by a G protein-coupled receptor and a proteinaccording to the above 1, which comprises:

a cell capable of expressing a protein according to the above 1; and

an oligonucleotide according to the above 31 or an antibody according tothe above 20.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a drawing substitute showing the expression profile of aninventive protein in human tissues;

FIG. 2 is a schematic view indicating the dopamine D1 receptorantagonistic effects of various test substance (Example 17);

FIG. 3 is a schematic view indicating the dopamine D1 receptor agonisticeffects of various test substance (Example 18);

FIG. 4 is a schematic view indicating the adenosine A2a receptorantagonistic effects of DMPX (Example 21);

FIG. 5 is a schematic view indicating the adenosine A2a receptoragonistic effects of CGS-21680 (Example 22);

FIG. 6 is a schematic view indicating that dopamine caused a signaltransduction mediated by the dopamine D1 receptor and the Gm1 (Example24); and

FIG. 7 is a schematic view indicating that adenosine caused a signaltransduction mediated by the adenosine A2a receptor and the Gm1 (Example24).

DESCRIPTION OF THE PREFERRED EMBODIMENTS <Inventive Proteins>

An inventive protein is a protein comprising any amino acid sequenceselected from the group consisting of:

(a) The amino acid sequence represented by SEQ ID NO:1;(b) An amino acid sequence of a protein involved in a G protein-coupledreceptor mediated signal transduction which consists of an amino acidsequence having a homology of 85% or more with the amino acid sequencerepresented by SEQ ID NO:1;(c) The amino acid sequence represented by SEQ ID NO:25;(d) The amino acid sequence represented by SEQ ID NO:26;(e) An amino acid sequence of a protein involved in a G protein-coupledreceptor mediated signal transduction which comprises the amino acidsequence of the amino acid Nos. 96 to 126 of SEQ ID NO:1;(f) An amino acid sequence of a protein involved in a G protein-coupledreceptor mediated signal transduction which comprises an amino acidsequence having a homology of 95% or more with the amino acid sequenceof the amino acid Nos. 96 to 126 of SEQ ID NO:1;(g) An amino acid sequence of a protein involved in a G protein-coupledreceptor mediated signal transduction which comprises at its N-terminalthe amino acid sequence of the amino acid Nos. 1 to 126 of SEQ ID NO:1;and(h) An amino acid sequence of a protein involved in a G protein-coupledreceptor mediated signal transduction which comprises at its N-terminalan amino acid sequence having a homology of 65% or more with the aminoacid sequence of the amino acid Nos. 1 to 126 of SEQ ID NO:1;

The inventive protein includes not only said protein but also its saltor derivative as long as its biological functions are not affected. Aderivative is not limited particularly and may for example be one whoseC terminal or other carboxyl group is converted into an amide, ester andthe like, or one whose N terminal or other amino group is protected forexample by a formyl group or acyl group. As a salt, an acid additionsalt is exemplified preferably. An acid addition salt may for example bea salt with an inorganic acid such as hydrochloric acid, phosphoricacid, sulfuric acid and the like, or a salt with an organic acid such asformic acid, acetic acid, propionic acid and the like.

<First Protein>

The first protein according to the invention is a protein (1) or (2)shown below.

(1) The protein consisting of the amino acid sequence represented by SEQID NO:1.(2) A protein involved in a G protein-coupled receptor mediated signaltransduction which consists of an amino acid sequence having a homologyof 85% or more with the amino acid sequence represented by SEQ ID NO:1.

The amino acid sequence represented by SEQ ID NO:1 comprises an aminoacid sequence part having a high homology with the GTP binding site andthe GTPase activation site conserved among G protein α subunits. Suchparts are the regions of the amino acid Nos. 126 to 133, 287 to 292, 353to 359, 428 to 435 in the amino acid sequence represented by SEQ IDNO:1. Any of these amino acids is in agreement with the GTP binding siteand the GTPase activation site of Gs and Golf which has already beenidentified as G protein α subunits (NATURE, 117-127, 1991, vol. 349).

Furthermore, the amino acid also comprises a sequence which is identicalto the characteristic sequence conserved highly in Gs and Golf proteinbelonging especially to the Gs family among the G protein α subunits(amino acid Nos. 119 to 126 in SEQ ID NO:1), and can also form an αhelix structure conserved among the G protein α subunits.

Based on these findings, the protein comprising the amino acid sequencerepresented by SEQ ID NO:1 is considered to be a G protein α subunit.

The fact that a protein (2) functions as a molecule involved in theintracellular signal transduction by a GPCR stimulation can be verifiedby means of a screening method according to the invention discussedbelow.

The amino acid sequence of a protein (2) preferably has a homology of90% or more, especially 95% or more with the amino acid sequencerepresented by SEQ ID NO:1.

An index indicating which and how many amino acid residues in a protein(2) can be substituted, deleted or added without losing any biologicalfunctions can be identified for example by a GTP binding level assaydescribed below. A variation causing no loss of the biological functionscan be conducted for example in a part having a low homology with theamino acid sequence of any of various G protein α subunits which havealready been identified.

In the case for example of an amino acid substitution, the amino acidcan be substituted by an amino acid having the characteristics similarto those of the former amino acid in terms of polarity, electric charge,solubility, hydrophilicity/hydrophobicity, polarity and the like, inview of the maintenance of the protein structure. In this context,glycine, alanine, valine, leucine, isoleucine and proline are classifiedinto non-polar amino acids; serine, threonine, cysteine, methionine,asparagine and glutamine are classified into polar amino acids;phenylalanine, tyrosine and triptophane are classified into aromaticside chain-carrying amino acids; lysine, arginine and histidine areclassified into basic amino acids; aspartic acid and glutamic acid areclassified into acidic amino acids. Accordingly, the substitution may beconducted using an amino acid selected from the same group.

A protein (2) also includes proteins derived from other speciescorresponding to the human protein. Such an other species-derivedcorresponding protein can be deduced from a nucleotide sequenceidentified by means for example of a screening of a gene library ofother species using a full length inventive polynucleotide or a partthereof as well as a 5′-RACE. Otherwise, a deductive identification ispossible also from a corresponding gene of other species screened by anNCBI Blast search described below,

A protein (2) may for example be the protein consisting of the aminoacid sequence represented by SEQ ID NO:25 and the protein consisting ofthe amino acid sequence represented by SEQ ID NO:26.

<Second Protein>

The second protein according to the invention is a protein (3) or (4)shown below.

(3) A protein involved in a G protein-coupled receptor mediated signaltransduction which comprises the amino acid sequence of the amino acidNos. 96 to 126 of SEQ ID NO:1.(4) A protein involved in a G protein-coupled receptor mediated signaltransduction which comprises an amino acid sequence having a homology of95% or more with the amino acid sequence of the amino acid Nos. 96 to126 of SEQ ID NO:1.

A protein (4) preferably has a homology of 96% or more, especially 97%or more with the amino acid Nos. 96 to 126 in the amino acid sequencerepresented by SEQ ID NO:1.

For the purpose of functioning as a molecule involved in anintracellular signal transduction by a GPCR stimulation, for example, asa G protein α subunit, each of the protein (3) and (4) usually has anamino acid sequence of the amino acid Nos. 75 to 133, 287 to 292, 353 to359 and 428 to 435 in the amino acid sequence represented by SEQ IDNO:1, or preferably has an amino acid sequence having a homology usuallyof 80% or more, especially 90% or more with the amino acid sequence ofthese regions. The total amino acid number is usually about 320 to 489,preferably about 350 to 460.

An index indicating which and how many amino acid residues in a protein(4) can be substituted, deleted or added without losing any biologicalfunctions of the protein (3) can be identified for example by a GTPbinding level assay described below. A variation causing no loss of thebiological functions can be conducted for example in a part having a lowhomology with the amino acid sequence of any of various G protein αsubunits which have already been identified. Also similarly to the firstprotein described above, the substitution of a base can be conducted sothat the amino acid obtained after a translation can possess thecharacteristics analogous to those of the amino acid before thesubstitution, with regard to polarity, electric charge, solubility,hydrophilicity/hydrophobicity, polarity and the like.

<Third Protein>

The third protein of the invention is a protein (5) or (6) shown below.

(5) A protein involved in a G protein-coupled receptor mediated signaltransduction which comprises at its N-terminal of the amino acidsequence of the amino acid Nos. 1 to 126 of SEQ ID NO:1.(6) A protein involved in a G protein-coupled receptor mediated signaltransduction which comprises at its N-terminal of an amino acid sequencehaving a homology of 65% or more with the amino acid sequence of theamino acid Nos. 1 to 126 of SEQ ID NO:1.

In a protein (6), the amino acid sequence part corresponding to the 126amino acid sequence at the N terminal of the protein (5) (hereinafterreferred to as a “specific N terminal amino acid sequence”) preferablyhas a homology of 70% or more, especially 75% or more with the specificN terminal amino acid sequence of the protein (5).

For the purpose of functioning as a molecule involved in anintracellular signal transduction by a GPCR stimulation, for example, asa G protein α subunit, each of the protein (5) and (6) usually has anamino acid sequence of the amino acid Nos. 75 to 133, 287 to 292, 353 to359 and 428 to 435 in the amino acid sequence represented by SEQ IDNO:1, or preferably has an amino acid sequence having a homology usuallyof 80% or more, especially 90% or more with these regions. The totalamino acid number is usually about 320 to 480, preferably about 350 to460.

An index indicating which and how many amino acid residues in a protein(6) can be substituted, deleted or added without losing any biologicalfunctions of the protein (5) can be identified for example by a GTPbinding level assay described below. A variation causing no loss of thebiological functions can be conducted for example in a part having a lowhomology with the amino acid sequence of any of various G protein αsubunits which have already been identified. Also similarly to the firstprotein described above, the substitution of a base can be conducted sothat the amino acid obtained after a translation can possess thecharacteristics analogous to those of the amino acid before thesubstitution, with regard to polarity, electric charge, solubility,hydrophilicity/hydrophobicity, polarity and the like.

There is no known G protein α subunit having the amino acid sequence ofthe amino acid Nos. 1 to 126 of the amino acid sequence represented bySEQ ID NO:1 or a sequence analogous thereto.

<Inventive Protein Production Method>

A protein of the invention can be produced by i) a method for separatinga membrane fraction containing said protein from a cell or tissue of ahuman or other animal species followed by a known protein purificationprocess, ii) a method employing a transformant of the inventiondescribed below or iii) a known chemical synthesis of a protein and thelike.

In a method i), a cell or tissue of a mammalian animal including a humancan be employed without limitation. It is particularly preferred to usea human cell or tissue, especially, a human brain-, uterus- orheart-derived cell or tissue.

A membrane fraction containing a protein of the invention can beobtained by suspending a cell or tissue for example in a HE/PI buffer(20 mM Hepes, 2 mM EDTA, 1× Proteinase inhibitor cocktail(Nacalaitesque)), pulverizing or lysing the suspension by means forexample of an ultrasonic treatment, homogenization, passage through aneedle of about 26 gauge, centrifuging the resultant pulverization orlysis solution at about 100 to 150×G for 5 to 10 minutes, centrifugingthe resultant supernatant at about 18,000 to 20,000 G for 20 to 30minutes, and then recovering the pellets.

The fact that the resultant cell membrane fraction contains a protein ofthe invention can be verified for example by a Western blotting using anantibody of the invention as described below.

A known protein purification method may for example be any of variouschromatographic procedure such as an ion exchange, gel filtration,affinity chromatography. As a method iii), a method described forexample in “The Peptide”, Academic Press, New York (1966) or a methodemploying a commercial protein synthesis resin may be exemplified.

It is also possible to obtain a protein (2) from a transformant having avariant DNA which is formed by imparting a DNA encoding the protein (1)with a variation using a known method such as one described in MolecularCloning: A Laboratory Manual, 2nd edition, Vol. 1 to 3, Cold SpringHarbor Laboratory Press (1989), Methods in Enzymology p 448 (1989), PCRA Practical Approach IRL Press p 200 (1991) and the like, for example, asite-specific mutation introduction, a PCR employing a variation primer.This may analogously be applied to a method for obtaining the protein(3) from a protein (4) and a method for obtaining the protein (6) from aprotein (5).

<Application of Inventive Proteins>

A protein of the invention can preferably be employed as a regulator ofa signal transduction mediated by a GPCR stimulation. Specifically, itcan preferably be employed for treating or preventing a diseaseassociated with an intracellular signal transduction relating to thedefect, reduced expression level or reduced function of a protein of theinvention.

<Inventive Polynucleotide>

A polynucleotide according to the invention is a polynucleotide encodinga protein of the invention described above, and comprises a nucleotidesequence selected from the group consisting of:

(a) A nucleotide sequence encoding the amino acid sequence representedby SEQ ID NO:1;(b) A nucleotide sequence encoding an amino acid sequence of a proteininvolved in a G protein-coupled receptor mediated signal transductionwhich consists of an amino acid sequence having a homology of 85% ormore with the amino acid sequence represented by SEQ ID NO:1;(c) A nucleotide sequence encoding the amino acid sequence representedby SEQ ID NO:25;(d) A nucleotide sequence encoding the amino acid sequence representedby SEQ ID NO:26;(e) A nucleotide sequence encoding an amino acid sequence of a proteininvolved in a G protein-coupled receptor mediated signal transductionwhich comprises the amino acid sequence of the amino acid Nos. 96 to 126of SEQ ID NO:1;(f) A nucleotide sequence encoding an amino acid sequence of a proteininvolved in a G protein-coupled receptor mediated signal transductionwhich comprises an amino acid sequence having a homology of 95% or morewith the amino acid sequence of the amino acid Nos. 96 to 126 of SEQ IDNO:1;(g) A nucleotide sequence encoding an amino acid sequence of a proteininvolved in a G protein-coupled receptor mediated signal transductionwhich comprises at its N-terminal the amino acid sequence of the aminoacid Nos. 1 to 126 of SEQ ID NO:1;(h) A nucleotide sequence encoding an amino acid sequence of a proteininvolved in a G protein-coupled receptor mediated signal transductionwhich comprises at its N-terminal an amino acid sequence having ahomology of 65% or more with the amino acid sequence of the amino acidNos. 1 to 126 of SEQ ID NO:1;(i) The nucleotide sequence represented by SEQ ID NO:2;(j) A nucleotide sequence encoding a protein involved in a Gprotein-coupled receptor mediated signal transduction which is anucleotide sequence having a homology of 85% or more with thepolynucleotide consisting of the nucleotide sequence represented by SEQID NO:2;(k) The nucleotide sequence represented by SEQ ID NO:27;(l) The nucleotide sequence represented by SEQ ID NO:28;(m) A nucleotide sequence encoding a protein involved in a Gprotein-coupled receptor mediated signal transduction which comprisesthe nucleotide sequence of the nucleotide Nos. 289 to 378 of SEQ IDNO:2;(n) A nucleotide sequence encoding a protein involved in a Gprotein-coupled receptor mediated signal transduction which comprises anucleotide sequence having a homology of 90% or more with thepolynucleotide consisting of the nucleotide sequence of the nucleotideNos. 289 to 378 of SEQ ID NO:2;(o) A nucleotide sequence encoding a protein involved in a Gprotein-coupled receptor mediated signal transduction which comprises atits 5′ terminal the nucleotide sequence of the nucleotide Nos. 1 to 378of SEQ ID NO:2; and(p) A nucleotide sequence encoding a protein involved in a Gprotein-coupled receptor mediated signal transduction which comprises atits 5′ terminal a nucleotide sequence having a homology of 70% or morewith the polynucleotide consisting of the nucleotide sequence of thenucleotide Nos. 1 to 378 of SEQ ID NO:2.

A polynucleotide of the invention (including an oligonucleotide)includes a polynucleotide comprising a nucleotide sequence describedabove and a polynucleotide complementary thereto. Unless otherwisespecified, a polynucleotide includes the both of a DNA and an RNA. A DNAincludes a single-stranded DNA having its nucleotide sequence, and asingle-stranded DNA complementary thereto, and a double-stranded DNA. ADNA, unless otherwise specified, includes a cDNA, genome DNA andsynthetic DNA. An RNA, unless otherwise specified, includes a total RNA,mRNA, rRNA and synthetic RNA.

An inventive polynucleotide is detailed with referring to the followingfirst, second and third polynucleotide described below.

<First Polynucleotide>

(7) The polynucleotide consisting of the nucleotide sequence representedby SEQ ID NO:2.(8) A polynucleotide encoding a protein involved in a G protein-coupledreceptor mediated signal transduction which consists of a nucleotidesequence having a homology of 85% or more with the polynucleotideconsisting of the nucleotide sequence represented by SEQ ID NO:2.

A polynucleotide (8) preferably has a homology of 87% or more,especially 90% or more with the polynucleotide (7).

A polynucleotide (8) is preferably one which hybridizes under astringent condition with the polynucleotide (7) consisting of thenucleotide sequence represented by SEQ ID NO:2. In the invention, astringent condition may for example be a condition involving 2×SSC,1×Denhart's solution at about 60° C.

An index indicating which and how many bases in a polynucleotide (8) canbe substituted, deleted or added without losing any biological functionsof the protein encoded by the polynucleotide (7) can be identified forexample by a cAMP assay or a GTP binding level assay described below. Avariation causing no loss of the biological functions can be conductedfor example in a part having a low homology with the polynucleotidesequence of any of various G protein α subunits which have already beenidentified.

Also similarly to the first protein described above, the substitution ofa base can be conducted so that the amino acid obtained after atranslation can possess the characteristics analogous to those of theamino acid before the substitution, with regard to polarity, electriccharge, solubility, hydrophilicity/hydrophobicity, polarity and thelike.

When a single amino acid has several translation codons, the basesubstitution within these translation codons may also be possible. Forexample, when alanine has 4 translation codons, namely, GCA, GCC, GCGand GCT, the third base in each codon can be substituted with each otheramong ATGC.

A polynucleotide (8) includes a polynucleotide of other speciescorresponding for example to a human polynucleotide. Such apolynucleotide can be screened for using NCBI blast search. Typically, anucleotide sequence containing the nucleotide residues 289 to 378 of SEQID NO:2 is subjected to an NCBI blast search to thereby search thenucleotide sequence database of other species and an EST database for asequence having a high homology. By screening the nucleotide sequencesselected by the search for a nucleotide sequence whose regioncorresponding to the nucleotide residues 289 to 378 has a homology forexample of 90% or more, a corresponding gene of other species can bescreened for.

A polynucleotide (8) is preferably one whose nucleotide sequencecorresponding to the nucleotide Nos. 1 to 222, 400 to 858, 877 to 1056,1078 to 1281 and 1306 to 1377 in SEQ ID NO:2 in the polynucleotide (7)has a homology usually of 75% or more, especially 80% or more with therespective nucleotide sequence of the polynucleotide (7).

A polynucleotide (8) may for example be a polynucleotide consisting ofthe nucleotide sequence represented by SEQ ID NO:27 and a polynucleotideconsisting of the nucleotide sequence represented by SEQ ID NO:28.

<Second Polynucleotide>

(9) A polynucleotide encoding a protein involved in a G protein-coupledreceptor mediated signal transduction which comprises the nucleotidesequence of the nucleotide Nos. 289 to 378 of SEQ ID NO:2.(10) A polynucleotide encoding a protein involved in a G protein-coupledreceptor mediated signal transduction which comprises a nucleotidesequence having a homology of 90% or more with the polynucleotideconsisting of the nucleotide sequence of the nucleotide Nos. 289 to 378of SEQ ID NO:2.

In a polynucleotide (10), the nucleotide sequence corresponding to thenucleotide Nos. 289 to 378 in SEQ ID NO:2 of a polynucleotide (9) has ahomology of 93% or more, especially 95% or more with the respectivesequence of (9).

A polynucleotide (10) preferably has a polynucleotide sequence whichhybridizes under a stringent condition with a polynucleotide consistingof the nucleotide sequence of the nucleotide Nos. 289 to 378 in SEQ IDNO:2.

Each of the protein (9) and (10), for achieving the function of theprotein encoded thereby as a molecule involved in an intracellularsignal transduction by a GPCR stimulation, for example, as a G protein αsubunit, usually has a nucleotide sequence of the nucleotide Nos. 223 to399, 859 to 876, 1057 to 1077, 1282 to 1305 in the nucleotide sequencerepresented by SEQ ID NO:2, or preferably has an nucleotide sequencehaving a homology usually of 85% or more, especially 90% or more withthese regions. The total nucleotide number is usually about 963 to 1443,especially 1053 to 1383.

An index indicating which and how many bases in a polynucleotide (10)can be substituted, deleted or added without losing any biologicalfunctions of the protein encoded by the polynucleotide (9) is similar tothat described above with regard to the first polynucleotide.

<Third Polynucleotide>

(11) A polynucleotide encoding a protein involved in a G protein-coupledreceptor mediated signal transduction which comprises at its 5′ terminalthe nucleotide sequence of the nucleotide Nos. 1 to 378 of SEQ ID NO:2.(12) A polynucleotide encoding a protein involved in a G protein-coupledreceptor mediated signal transduction which comprises at its 5′ terminala nucleotide sequence having a homology of 70% or more with thepolynucleotide consisting of the nucleotide sequence of the nucleotideNos. 1 to 378 of SEQ ID NO:2.

In a polynucleotide (12), the nucleotide sequence part corresponding tothe 378 nucleotide sequence at the 5′ terminal of the protein (11)(hereinafter referred to as a “specific 5′ terminal nucleotidesequence”) preferably has a homology of 75% or more, especially 80% ormore with the specific 5′ terminal amino acid sequence of (11).

A polynucleotide (12) preferably has at its 5′ terminal a polynucleotidesequence which hybridizes under a stringent condition with thepolynucleotide consisting of the nucleotide sequence of the nucleotideNos. 1 to 378 in SEQ ID NO:2.

Each of the protein (11) and (12), for achieving the function of theprotein encoded thereby as a molecule involved in an intracellularsignal transduction by a GPCR stimulation, for example, as a G protein αsubunit, usually has a nucleotide sequence of the nucleotide Nos. 223 to399, 859 to 876, 1057 to 1077, 1282 to 1305 in the nucleotide sequencerepresented by SEQ ID NO:2, or preferably has an nucleotide sequencehaving a homology usually of 85% or more, especially 90% or more withthese regions. The total nucleotide number is usually about 963 to 1443,especially 1053 to 1383.

An index indicating which and how many bases in a polynucleotide (12)can be substituted, deleted or added without losing any biologicalfunctions of the protein encoded by the polynucleotide (11) is similarto that described above with regard to the first polynucleotide.

There is no polynucleotide encoding a G protein α subunit having thenucleotide sequence of the nucleotide Nos. 1 to 378 in SEQ ID NO:2 or asequence analogous thereto.

<Inventive Polynucleotide Production Method>

Polynucleotides (7) to (12) can be obtained for example by a screening ahuman DNA library by a hybridization using as a probe an oligonucleotide(for example, an oligonucleotide of the invention described below)synthesized based on the nucleotide sequence represented by SEQ ID NO:2.They can be obtained also by a PCR in a standard manner after preparingsuitable primers (for example, oligonucleotides of the inventiondescribed below) based for example on the nucleotide sequence of SEQ IDNO:2 using as a PCR template a cDNA library for example of a human, ratand mouse. They can be obtained also by a chemical synthesis.

As a cDNA library, one derived from a brain, thymus, testes, spleen,small intestine, uterus and heart is preferred.

A method for obtaining a polynucleotide (10) by introducing a variationinto a polynucleotide (9) and a method for obtaining a polynucleotide(12) by introducing a variation into a polynucleotide (11) are asdescribed above.

<Application of Inventive Polynucleotide>

An inventive polynucleotide can be used preferably as a regulator of anintracellular signal transduction mediated by a GPCR stimulation.Typically, it can be used preferably for treating or preventing adisease caused by an abnormality in this intracellular signaltransduction. It is useful especially in treating or preventing adisease associated with an intracellular signal transduction relating tothe defect, reduced expression level or reduced function of a protein ofthe invention.

An inventive polynucleotide can be used preferably also in screening fora substance capable of regulating a signal transduction mediated by aGPCR and a G protein of the invention.

<Inventive Recombinant Vector and Transformant>

An inventive recombinant vector is a vector containing an inventivepolynucleotide (which herein is a DNA). For example, it may be a vectorcapable of expressing a protein of the invention.

A vector capable of expressing a protein of the invention can beproduced by ligating an inventive polynucleotide to an expressibleposition downstream of a promoter of an expression vector in accordancewith a standard method.

An expression vector may be selected from known vectors capable ofreplicating in host cells as appropriate depending on the host cells.For example, a pBR322, pUC12, pUC119 and pBluescript can be exemplifiedwhen an E. coli is employed as a host cell, while a pUB110 and pC194 areexemplified when a Bacillus organism is employed as a host cell. An Yip5and Yep24 are exemplified when using an yeast as a host cell. An AcNPVis exemplified when using an insect cell as a host cell. A pUC18 andpUC19 are exemplified when using an animal cell as a host cell.

A host cell may be any of those known in the art without limitation.Those which may be exemplified are bacteria such as an E. coli (forexample, K12) and a Bacillus microorganism (for example, MI114), yeasts(for example, AH22), insect cells (for example, Sf cell), animal cells(for example, COS-7 cell, Vero cell, CHO cell and the like).

A method for transforming a host cell with an inventive recombinantvector may be a known method selected suitably depending on the hostcell. A known introduction method may for example be a calcium phosphatemethod, electroporation, lipofection, DEAE dextran method and the like.From transformants, an inventive transformant is selected for example bymeans of a drug resistance marker possessed by the vector as an index.

<Inventive Protein Production Method>

A method for producing a protein of the invention is a method in whichan inventive transformant is cultured and an inventive protein isrecovered from the resultant culture product.

The conditions of culturing a transformant may be selected appropriatelydepending on the type of the transformant.

When an inventive transformant is a microorganism, the culture isconducted in a liquid medium or plate medium employed usually forculturing a microorganism. The culture temperature may be within therange allowing a microorganism to be grown, for example from 15 to 40°C. The culture medium pH may also be within the range allowing amicroorganism to be grown, for example about pH6 to 8. The culture timeperiod may vary depending on other culturing conditions, and may usuallybe 1 to 5 days, especially 1 to 2 days. When using an inducibleexpression vector such as those of the temperature shift type or IPTGinducible type, the induction time period may be within a day,especially within several hours.

Also when an inventive transformant is a mammalian cell, it can becultured under the condition suitable for said cell. For example, anFBS-supplemented DMEM medium (NISSUI) may be employed to conduct aculture in the presence of 5% CO₂ at a temperature of 36 to 38° C. withreplacing the medium with a fresh one at an interval of several days.Upon confluent growth, the cells were combined with a trypsin PBSsolution to disperse into individual cells and the resultant cellsuspension was diluted by several times and inoculated onto a new petridish, which is then subjected to a subculture. The culture time periodis usually 2 to 5 days, especially 2 to 3 days.

Also when an insect cell is employed as a transformant, the culturecondition may be adjusted appropriately depending on the type of thecell. For example, an insect cell culture medium such as Grace's mediumcontaining FBS and Yeastlate may be employed to conduct the culture at25 to 35° C. The culture time period is 1 to 5 days, especially 2 to 3days. When using as a vector an virus-containing transformant such as aBaculovirus, the culture is continued preferably until the time beforethe cell death as a result of the onset of the cytoplasmic effect (forexample, 3 to 7 days, especially 4 to 6 days).

After completion of the culture, the transformant cells were recoveredby a centrifugation, suspended in a suitable buffer if desired, anddispersed by means of a polytron, ultrasonic treatment, homogenizer andthe like. The resultant dispersion is centrifuged at about 100 to 150 Gfor about 5 to 10 minutes, and the resultant supernatant is centrifugedat about 18,000 to 20,000 G for about 20 to 30 seconds to recover thepellet, whereby obtaining a cell membrane fraction.

The cell membrane fraction thus obtained is subjected to a known proteinpurification method, such as any of chromatographic methods includingion exchange, hydrophobic, gel filtration and affinity chromatographies,whereby purifying the protein according to the invention.

An inventive protein can be expressed for example as being attached witha histidine tag, or as a glutathion S transferase fusion protein. Theformer case employs a metal chelate affinity column, while the lattercase employs a glutathion S transferase monoclonal antibody column,whereby accomplishing the purification of an inventive protein in afurther convenient manner.

<Inventive Antisense Polynucleotide> <Aspect>

An inventive antisense polynucleotide is a polynucleotide (13) or (14)shown below.

(13) A polynucleotide which inhibits expression of a protein of theinvention which consists of a nucleotide sequence complementary to atleast 15 contiguous nucleotides in the nucleotide sequence representedby SEQ ID NO:2.(14) A polynucleotide which inhibits expression of a protein of theinvention which hybridizes under an intracellular condition with apolynucleotide consisting of at least 15 contiguous nucleotides in thenucleotide sequence represented by SEQ ID NO:2.

An antisense oligonucleotide of the invention hybridizes with a mRNAencoding an inventive protein to inhibit the translation from the mRNAto the protein or cleaves the mRNA, whereby inhibiting the expression ofthis protein.

While the upper limit of the number of the nucleotides in an antisensepolynucleotide is not limited particularly, it is usually about 60nucleotides for the purpose of achieving the objective.

A polynucleotide (13) preferably has a nucleotide sequence complementaryto at least 30 nucleotides, especially at least 50 nucleotides in thepolynucleotide represented by SEQ ID NO:2. An antisense (14) ispreferably one which hybridizes under an intracellular condition alsowith a polynucleotide consisting of at least 30 nucleotides, especiallyat least 50 nucleotides in the polynucleotide represented by SEQ IDNO:2.

A polynucleotide which hybridizes under an intracellular condition inthe invention may for example be a polynucleotide which hybridizes undera stringent condition described below. The stringent condition may forexample be a condition involving 2×SSC, 1×Denhart's solution at about60° C.

A polynucleotide (13) preferably has a nucleotide sequence complementaryto at least 15 contiguous nucleotides across the both of the region ofthe nucleotide Nos. 1 to 378 and the region of the nucleotide Nos. 379to 1377 in SEQ ID NO:2.

Similarly, a polynucleotide (14) is preferably one which hybridizesunder an intracellular condition with a polynucleotide consisting of atleast 15 contiguous nucleotides across the both of the region of thenucleotide Nos. 1 to 378 and the region of the nucleotide Nos. 379 to1377 in SEQ ID NO:2.

“At least 15 contiguous nucleotides in the nucleotide sequencerepresented by SEQ ID NO:2” in polynucleotides (13) and (14) which iscloser to the 5′ terminal of SEQ ID NO:2 is more preferable.

An antisense polynucleotide of the invention may be a single-strandedDNA, double-stranded DNA, single-stranded RNA, double-stranded RNA orDNA-RNA hybrid. When a double-stranded RNA is employed, it is generallycalled an RNAi. A derivative of such a nucleotide may also be employedas long as it inhibits the expression of an inventive protein. Aderivative may for example be a phosphorthioate DNA, H-phosphonate DNAand the like.

Inhibition of an inventive protein expression by an inventive antisensepolynucleotide can be verified for example by a method described below.A human brain-derived cell is combined with an antisense at about 5 nMto 10 μM if necessary together with a known intracellular introductionreagent such as a lipofection reagent, lipofectamine reagent, liposomeand the like. Then, from this cell a cell extract is prepared by a knownmethod and an inventive antibody is used to measure expression level ofthe inventive protein by a known method such as an ELISA or westernblotting. This expression level is compared with the level observed inthe absence of the antisense.

An inventive antisense polynucleotide may for example be one whichreduces the inventive protein expression level for example to 70% orless, preferably to 50% or less based on the level in the absence of theantisense polynucleotide.

An inventive antisense polynucleotide can be produced by a knownchemical synthesis method.

<Application of Antisense Polynucleotide>

An inventive antisense polynucleotide can be used preferably as aregulator of an intracellular signal transduction mediated by a GPCRstimulation. Typically, it can be used preferably for treating orpreventing a disease caused by an abnormality in this intracellularsignal transduction. It is useful preferably for the purpose especiallyof suppressing the increased abnormality in this intracellular signaltransduction.

<Ribozyme> <Aspect>

An inventive ribozyme is a ribozyme (15) or (16) shown below.

(15) A ribozyme having an ability of cleaving a polynucleotide of theinvention which comprises two polynucleotide regions complementary totwo regions respectively consisting of at least 9 contiguous nucleotideswhich are two regions in the nucleotide sequence represented by SEQ IDNO:2.(16) A ribozyme having an ability of cleaving a polynucleotide of theinvention which comprises two polynucleotide regions which hybridizesunder an intracellular condition with two regions respectivelyconsisting of at least 9 contiguous nucleotides which are two regions inthe nucleotide sequence represented by SEQ ID NO:2.

A ribozyme (15) preferably comprises two polynucleotide regionscomplementary to two regions respectively consisting of at least 10,especially 11 contiguous nucleotides which are two regions in thenucleotide sequence represented by SEQ ID NO:2.

A ribozyme (16) preferably comprises two polynucleotide regions whichhybridizes under an intracellular condition with two regionsrespectively consisting of at least 10, especially 11 contiguousnucleotides which are two regions in the nucleotide sequence representedby SEQ ID NO:2.

In the ribozymes (15) and (16), the two regions in the nucleotidesequence represented by SEQ ID NO:2 may be adjacent to each other, orbut may preferably be interrupted by about 1 to 4 nucleotides presentbetween them. For example, a hammer-head ribozyme may contain a singleinterrupting nucleotide, while a hairpin ribozyme may contain 4interrupting nucleotides.

A ribozyme is an RNA molecule containing an antisense sequencerecognizing a specific site of an RNA, and has an RNA cleavage enzymeactivity. As a result, the ribozyme recognizes its target RNA, andcleaves a certain site of the RNA specifically.

An inventive ribozyme may be of a hammer-head or hairpin type. Ahammer-head type usually recognize an NUX (N is G, U, C or A, while X isC, U or A) and cleaves a mRNA at the 3′-position of the X.

A hammer-head ribozyme according to the invention may for example be aribozyme comprising a nucleotide sequence listed below.

A ribozyme comprising the nucleotide sequence:

(SEQ ID NO:3) UCGCCUCCUUCUGAUGAGGCCGAAAGGCCGAAACCGCCTCGCGC.The ribozyme having this nucleotide sequence once forms its conformationand then recognizes the nucleotide sequence of the nucleotide Nos. 273to 295 in SEQ ID NO:2.A ribozyme comprising the nucleotide sequence:

(SEQ ID NO:4) CGGCCGCCCGCUGAUGAGGCCGAAAGGCCGAAACUGGGGCCAGC.The ribozyme having this nucleotide sequence once forms its conformationand then recognizes the nucleotide sequence of the nucleotide Nos. 111to 133 in SEQ ID NO:2.A ribozyme comprising the nucleotide sequence:

(SEQ ID NO:5) CAGCGGCCGCCUGAUGAGGCCGAAAGGCCGAAACUGUAGCACA.The ribozyme having this nucleotide sequence once forms its conformationand then recognizes the nucleotide sequence of the nucleotide Nos. 8 to30 in SEQ ID NO:2.

A hairpin type ribozyme usually recognizes an NNNG/CN*GUCNNNNNNNN(N isG, U, C or A), and cleaves a mRNA between N*G.

A hairpin ribozyme according to the invention may for example be aribozyme comprising a nucleotide sequence listed below.

A ribozyme comprising the nucleotide sequence:UCGCCUCCUUAGAAGCCUACCAGAGAAACACACGUUGUGGUAUAUUACCUGGUA (SEQ ID NO:6).The ribozyme having this nucleotide sequence once forms its conformationand then recognizes the nucleotide sequence of the nucleotide Nos. 287to 295 in SEQ ID NO:2.A ribozyme comprising the nucleotide sequence:CGGCCGCCCGAGAAGGGGACCAGAGAAACACACGUUGUGGUAUAUUACCUGGUA (SEQ ID NO:7).The ribozyme having this nucleotide sequence once forms its conformationand then recognizes the nucleotide sequence of the nucleotide Nos. 116to 133 in SEQ ID NO:2.A ribozyme comprising the nucleotide sequence:CAGCGGCCGCAAGAAGUAGACCAGAGAAACACACGUUGUGGUAUAUUACCUGGUA (SEQ ID NO:8).The ribozyme having this nucleotide sequence once forms its conformationand then recognizes the nucleotide sequence of the nucleotide Nos. 12 to30 in SEQ ID NO:2.

While an inventive ribozyme usually consists of an RNA, a one whichincludes a deoxyribonucleotide or a derivative such as a phosphorthioateDNA which is difficult to be decomposed in vivo are also included in theinventive ribozyme.

An inventive ribozyme can be produced by a known chemical synthesismethod, in vitro or in vivo transcription and the like. Typically, an invitro transcription involves the ligation of a DNA having a sequencecomplementary to the sequence of SEQ ID NO:3, 4, 5, 6, 7 or 8 to thedownstream of the DNA encoding a promoter such as T7, T3 or SP6. Usingthis DNA as a template, a transcription reaction by an RNA polymerase isconducted. The resultant transcription product can be used as an RNA forthe ribozyme.

An in vivo transcription involves the integration of a DNA having asequence complementary to the sequence of SEQ ID NO:3, 4, 5, 6, 7 or 8into an mammalian expression vector followed by the transduction of thisexpression vector into a mammalian cell. As a result of the cellulartranscription mechanism, an RNA having the sequence of SEQ ID NO:3, 4,5, 6, 7 or 8 is synthesized.

<Application of Ribozyme>

An inventive ribozyme can be used preferably as a regulator of anintracellular signal transduction mediated by a GPCR stimulation.Typically, it can be used preferably for treating or preventing adisease caused by an abnormality in this intracellular signaltransduction. It is useful preferably for the purpose especially ofsuppressing the increased abnormality in this intracellular signaltransduction.

<Inventive Antibody> <Aspect>

An inventive antibody is an antibody which recognizes an inventiveprotein specifically. The inventive antibody may be a polyclonalantibody or monoclonal antibody. The inventive antibody includes anantibody having an antigen binding ability toward a polypeptideconsisting of 5 contiguous amino acids, preferably 10 amino acids in theamino acid sequence constituting an inventive protein. In addition, aderivative of such an antibody (chimera antibody and the like) or a onelabeled with an enzyme such as a peroxidase are also included in theinventive antibody.

<Method for Producing Inventive Antibody>

Any of the antibodies described above can be produced in accordance witha known production method (for example, Current protocols in MolecularBiology edit. Ausubel et al. (1987) Publish. John Wiley and Sons.Section 11.12-11.13).

Typically, when an inventive antibody is a polyclonal antibody, it canbe obtained by immunizing a non-human animal such as a rodent animalwith an inventive protein followed by the isolation from the serum ofthis immunized animal in accordance with a standard method. When aninventive antibody is a monoclonal antibody, it can be obtained from ahybridoma produced by immunizing a non-human animal such as a mouse witha polypeptide having an inventive protein or its partial sequencefollowed by fusing the spleen cell of this immunized animal with amyeloma cell (Current protocols in Molecular Biology edit. Ausubel etal. (1987) Publish. John Wiley and Sons. Section 11.4-11.11).

<Application of Antibody>

An inventive antibody can be used preferably as a regulator of anintracellular signal transduction mediated by a GPCR stimulation.Typically, it can be used preferably for treating or preventing adisease caused by an abnormality in this intracellular signaltransduction. It is useful preferably for the purpose especially ofsuppressing the increased abnormality in this intracellular signaltransduction.

An inventive antibody can preferably be used also in the affinitychromatography for purifying an inventive protein as well as in thescreening for a substance which may affect the expression of aninventive protein.

<Inventive Oligonucleotide>

An inventive oligonucleotide is an oligonucleotide (17) or (18) shownbelow.

(17) An oligonucleotide capable of recognizing a polynucleotiderepresented by SEQ ID NO:2 specifically which consists of at least 17contiguous nucleotides in the nucleotide sequence represented by SEQ IDNO:2.(18) An oligonucleotide capable of recognizing a polynucleotiderepresented by SEQ ID NO:2 specifically which has a homology of 80% ormore with at least 17 contiguous nucleotides in the nucleotide sequencerepresented by SEQ ID NO:2.

The length of each of the oligonucleotides (17) and (18) can be selectedappropriately depending on the use. It may be the full length of SEQ IDNO:2.

An oligonucleotide (18) preferably has a homology of 85% or more,especially 90% or more with an oligonucleotide (17).

The expression “recognize specifically” in conjunction witholigonucleotides (17) and (18) means that each oligonucleotide can beemployed to detect the nucleotide sequence represented by SEQ ID NO:2specifically or selectively by a known nucleotide sequencing means suchas a northern blotting or PCR.

An inventive oligonucleotide can be used as a probe or primer which candetect or amplify an RNA generated as a result of the expression of aninventive DNA or a polynucleotide derived therefrom in a specificmanner. Typically, it can be used as a probe or primer in a known methodfor detecting a certain nucleotide sequence, such as a Northernblotting, in situ hybridization or PCR.

As a result, the absence or presence of the expression of, or theexpression level of an inventive polynucleotide can be assessed.Accordingly, an inventive oligonucleotide can preferably used fordiagnosing a disease caused by a signal transduction abnormalityresulting from the defect of and the abnormal increase or decrease inthe expression level of the inventive protein. A test sample may be atotal RNA prepared by a standard method from a sample taken from atissue of a subject such as an uterus or any of various polynucleotidesprepared from such an RNA.

An inventive oligonucleotide may for example be ones having thenucleotide sequences of

(SEQ ID NO:9) 5′-ATGGGTCTGTGCTACAGTCTGCGG and (SEQ ID NO:10)5′-ACGATGGTGCTTTTCCCAGACTCACCAGCCCCGAGCA.This oligonucleotide set can preferably used as PCR primers foramplifying a protein of the invention.

<Inventive Screening Method>

A polynucleotide of the invention can be used for screening for asubstance which activates or inhibits (or suppress) the cellular signaltransduction mediated by a GCPR and an inventive protein.

A GPCR stimulating signal is transmitted to an effector via theactivation of a G protein as a result of the GDP/GTP exchange reactionon a G protein α subunit. Accordingly, by using the change in theactivity of this effector as an index in screening test substances, asubstance capable of activating or inhibiting the signal transductionmediated by a GPCR and a G protein α subunit can be identified. Inaddition, also by using the change in the level of the binding of a GTPto a membrane fraction of a cell expressing a G protein as an index inscreening test substances, a substance capable of activating orinhibiting the signal transduction mediated by a GPCR and a G protein αsubunit can be identified.

Otherwise, by screening test substances which alter the level of theexpression of an inventive protein, a substance capable of activating orinhibiting the signal transduction mediated by a GPCR and a G protein αsubunit can be identified.

<First Method>

A method for screening for a substance capable of regulating a signaltransduction mediated by a GPCR and an inventive protein comprising:

(a) a step for bringing a test substance into contact with a test cellhaving a recombinant vector containing an inventive polynucleotide(which herein is a DNA encoding an inventive protein) and a recombinantvector containing a DNA encoding a GPCR;(b) a step for measuring the G protein effector activity or the indexvalue correlating therewith in the test cell (hereinafter abbreviated as“effector activity”); and(c) a step for comparing this effector activity with the effectoractivity in the test cell which has not been brought into contact withthe test substance, whereby selecting a test substance capable ofaltering the effector activity in the test cell.

In the first method, an agonist of GPCR can be selected.

A cell in which a recombinant vector having a DNA encoding an inventiveprotein and a recombinant vector having a DNA encoding a GPCR arecontained may for example be but not limited to a mammalian cell orinsect cell. A mammalian cell may be any known cell such as a Vero cell,Hela cell, CV1 cell, COS1 cell, CHO cell and the like, which may beemployed without limitation. An insect cell may be any known cell suchas a Sf cell, MG1 cell, High Five™ cell, and the like, which may beemployed without limitation. The type of the vector is not limitedparticularly, and any known vector may be selected depending on the typeof the cell.

A DNA encoding a GPCR can be obtained by a method for screening a humancDNA library using a probe designed based on the nucleotide sequencesdescribed in a GPCR database (www.cmbi.kun.nl/7tm/), by a method forconducting a PCR using as a template a human cDNA library together withthe primers designed based on the nucleotide sequences described above,or by a chemical synthesis method and the like.

An effector of a G protein may be an effector which is a target of a Gprotein α subunit or may be an effector which is a target of a G proteinβγ subunit. It is also possible to measure an index value correlatingwith the effector activity of a G protein.

An effector which is targeted by a G protein α subunit may for examplebe an adenylate cyclase, Ca²⁺ channel, K⁺ channel, phospholipase Cβ andthe like. The adenylate cyclase activity be assessed by measuring theintracellular cAMP level. The Ca²⁺ channel activity can be assessed bymeasuring the cell membrane electric potential. The K⁺ channel activitycan be assessed by measuring the cell membrane electric potential. Thephospholipase Cβ activity can be assessed by measuring the Ca²⁺ level.

An effector which is targeted by a G protein βγ subunit may for examplebe an adenylate cyclase, Ca²⁺ channel, K⁺ channel, phospholipase Cβ,phosphatidyl inositol 3-kinase β or γ and the like. The phosphatidylinositol 3-kinase β or γ activity can be assessed by measuring the Ca²⁺level.

An effector to be examined for its activity is preferably an effectortargeted by a G protein α subunit, with an adenylate cyclase being morepreferred. The intracellular cAMP level which reflects the adenylatecyclase activity can be measured by a known method such as an RIAemploying an anti-cAMP antibody obtained by immunizing a mouse, rat,rabbit, goat, cattle and the like together with a ¹²⁵I-labeled cAMP,other EIA employing a combination of an anti-cAMP antibody and a labeledcAMP, a SPA method employing a scintillant obtained by immobilizing ananti-cAMP antibody using a protein A or an antibody against an animalIgG used for producing an anti-cAMP antibody together with a¹²⁵I-labeled cAMP, an EFC method employing a combination of an anti-cAMPantibody, enzyme donor-binding cAMP and enzyme blank acceptor, and thelike. Any of these measurements can be accomplished using a commercialkit.

An intracellular cAMP level can be assessed also by a method in which,for example, a CRE (cAMP response element, which reacts with acAMP)-containing DNA is inserted into the upstream of the reporter geneof a reporter gene vector to form a CRE-reporter gene vector, and thisvector is also introduced into a test cell and then the reporter geneexpression level is measured. In a cell into which a CRE-reporter genevector has been introduced, a stimulation accompanied with an elevationin the cAMP level induces a reporter gene expression mediated by the CREand the subsequent reporter protein production. On the contrary, areduction in the cAMP level leads to a reduction in the CRE-mediatedreporter protein production. By using a CRE reporter gene vector, thecAMP level can be measured conveniently at a high sensitivity.

As a reporter gene, any of known genes can be employed withoutlimitation, including a luciferase gene, secretor alkaline phosphatase(SEAP) gene, chloramphenicol acetyltransferase (CAT) gene,β-galactosidase gene and the like. Any of these genes can be examinedfor its expression level using a commercial measurement kit describedbelow. The luciferase gene expression level can be measured by adding aluminescent substrate luciferrin (manufactured for example by TOYO INK)to a cell solution followed by measuring the luminescence resulting fromthe decomposition of the substrate using a luminometer, liquidscintillation counter or top counter. Expression level of the alkalinephosphatase gene can be determined for example by using LμMi-Phos530(WAKO PURE CHEMICAL). Expression level of the chloramphenicolacetyltransferase gene can be determined using a FAST CATChloramphenicol Acetyltransferase Assay Kit (WAKO PURE CHEMICAL).Expression level of the β-galactosidase gene can be determined using anAURORA Gal-XE (WAKO PURE CHEMICAL).

In the case for example where a luciferase gene is employed, aCRE-containing DNA is inserted into a multiple cloning site in theupstream of a luciferase gene such as a PICK-A-GENE Basic Vector or aPICK-A-GENE Enhancer vector (TOYO INK) and the like, which is then usedas a CRE reporter gene vector.

The type of a test substance is not limited particularly. Those whichmay be exemplified are proteins, peptides, non-peptide compounds(nucleotides, amines, saccharides, lipids and the like), organic lowmolecular compounds, inorganic low molecular compounds, fermentationproducts, cell extracts, plant extracts, animal tissue extracts and thelike.

The contact of a cell with a test substance may be effected under acondition avoiding the cell death and allowing an inventive protein anda GPCR to be expressed from an introduced vector (temperature, pH,medium composition). The concentration of a test substance upon contactwith a cell may for example be about 0.001 to 10 μM, although it mayvary depending on the type of the substance.

A test substance which increase the effector activity of a test cellbrought into contact with the test substance for example by about 25%,preferably about 50%, more preferably about 100%, when compared with theeffector activity in the test cell which was not brought into contactwith the test substance, can be selected as a GPCR agonist.

<Second Method>

A method for screening for a substance capable of regulating a signaltransduction mediated by a GPCR and an inventive protein comprising:

(a) a step for bringing a test substance into contact with a test cellhaving a recombinant vector containing an inventive polynucleotide(which herein is a DNA encoding an inventive protein) and a recombinantvector containing a DNA encoding a GPCR;(b) a step for measuring the G protein effector activity in the testcell; and(c) a step for comparing this effector activity with the effectoractivity when the said test substance has been brought into contact witha control cell having no recombinant vector containing a DNA encoding aninventive protein but having a recombinant vector containing a DNAencoding a GPCR, whereby selecting a test substance causing a differencein the effector activity between the test cell and the control cell.

In the second method, a test substance which gives the effector activityof a test cell having an inventive protein expression vector which ishigher than the effector activity in a control cell having no such avector may be searched for. As a result, a substance which activates anystage of the signal transduction mediated by a GPCR and the inventiveprotein can be selected as a candidate compound.

A test substance which increase the effector activity of a test cell forexample by about 20%, preferably about 50%, more preferably about 100%,when compared with a control cell can be selected as a signaltransduction activator.

Otherwise, the aspect is similar to that in of the first methoddiscussed above.

<Third Method>

A method for screening for a substance capable of regulating a signaltransduction mediated by a GPCR and an inventive protein comprising:

(a) a step for bringing a test substance into contact with a test cellhaving a recombinant vector containing an inventive polynucleotide(which herein is a DNA encoding an inventive protein) and a recombinantvector containing a DNA encoding a GPCR;(b) a step for measuring the G protein effector activity in the testcell; and(c) a step for comparing this effector activity with the effectoractivity when the said test substance has been brought into contact witha control cell having no recombinant vector containing a DNA encoding aGPCR but having a recombinant vector having a DNA encoding an inventiveprotein, whereby selecting a test substance causing a difference in theeffector activity between the test cell and the control cell.

In the third method, a test substance which gives the effector activityof a test cell having a GPCR expression vector which is higher than theeffector activity in a control cell having no GPCR expression vector maybe searched for. As a result, a GPCR agonist can be selected as acandidate substance.

A test substance which increase the effector activity of a test cell forexample by about 20%, preferably about 50%, more preferably about 100%,when compared with a control cell can be selected as a signaltransduction activator.

Otherwise, the aspect is similar to that in of the first methoddiscussed above.

<Fourth Method>

A method for screening for a substance capable of regulating a signaltransduction mediated by a GPCR and an inventive protein comprising:

(a) a step for bringing a test substance and a GPCR ligand into contactwith a test cell having a recombinant vector containing an inventivepolynucleotide (which herein is a DNA encoding an inventive protein) anda recombinant vector containing a DNA encoding a GPCR protein;(b) a step for measuring the G protein effector activity in the testcell; and(c) a step for comparing this effector activity with the effectoractivity in the test cell which has not been brought into contact withthe test substance but has been brought into contact with the ligand,whereby selecting a test substance capable of altering the effectoractivity in the test cell.

In the fourth method, a test substance which gives increased or reducedeffector activity in the control cell which has not been brought intocontact with the test substances when compared with the effectoractivity in the test cell which has been brought into contact with thetest substances may be searched for. As a result, a substance whichactivates or inhibits any stage of the signal transduction initiatedfrom the binding of a GPCR ligand to a GPCR can be selected, including aGPCR agonist or antagonist.

A GPCR ligand may for example be amine molecules. It is preferableespecially to use dopamine. The ratio between a ligand to be broughtinto contact with a cell and a test substance, when represented as themolar ratio of ligand:test substance, may for example be about 1:0.1 to1:100, preferably about 1:1 to 1:50.

The percentage effector activity in a test cell which has been broughtinto contact with the both of a GPCR ligand and a test substance iscalculated, on the bases of the effector activity in the test cell whichhas been brought into contact only with the GPCR ligand being regardedas 100% and the effector activity in the test cell which has beenbrought into contact with none of the GPCR ligand or the test substanceas 0%. A test substance which gives a % effector activity in a test cellwhich has been brought into contact with the both of a GPCR ligand andthe test substance of 85% or less, preferably 70% or less, especially50% or less can be selected as a candidate of the cellular signaltransduction inhibitor or suppressor. On the other hand, a testsubstance which raises this percentage to 125% or more, preferably 150%or more, especially 200% or more can be selected as a candidate of thecellular signal transduction activator.

Otherwise, the aspect is similar to that in of the first methoddiscussed above.

<Fifth Method>

A method for screening for a substance capable of regulating a signaltransduction mediated by a GPCR and an inventive protein comprising:

(a) a step for bringing a test substance and a GPCR ligand into contactwith a test cell having a recombinant vector containing an inventivepolynucleotide (which herein is a DNA encoding an inventive protein) anda recombinant vector containing a DNA encoding a GPCR protein;(b) a step for measuring the G protein effector activity in the testcell;(c) a step for comparing this effector activity with the effectoractivity in the test cell which has not been brought into contact withthe test substance but has been brought into contact with the ligand,whereby investigating the change in the effector activity in the testcell; and(d) a step for comparing the rate of change in this effector activitywith the rate of change in the effector activity when the said testsubstance and said ligand has been brought into contact with a controlcell having no recombinant vector containing a DNA encoding a GPCR buthaving a recombinant vector containing a DNA encoding an inventiveprotein, whereby selecting a test substance causing a difference in therate of change in the effector activity between the test cell and thecontrol cell.

In the fifth method, a test substance which gives an elevated rate ofchange in the effector activity in a test cell having a recombinantvector containing a DNA encoding a GPCR protein when compared with therate of change in the effector activity in a control cell having no sucha vector may be searched for. As a result, a substance which serves asan antagonist against an exogenous GPCR can be selected. While in thefourth method described above an antagonist against an endogenous GPCRis also selected, by subjecting the substances obtained by the fourthmethod to a screening by the fifth method, a substance serving as anantagonist against the endogenous GPCR can selectively be eliminated.

A test substance which increase the rate of change in the effectoractivity of a test cell for example by about 15%, preferably about 30%,more preferably about 50%, when compared with a control cell can beselected as a candidate of an exogenous GPCR-directed antagonist.

Otherwise, the aspect is similar to that in of the first methoddiscussed above.

<Sixth Method>

A method for screening for a substance capable of regulating a signaltransduction mediated by a GPCR and an inventive protein comprising:

(a) a step for bringing a test substance into contact with a cellmembrane fraction of a cell having a recombinant vector containing aninventive polynucleotide (which herein is a DNA encoding an inventiveprotein) and a cell membrane fraction of a cell having a recombinantvector containing a DNA encoding a GPCR, ora cell membrane fraction of a cell having the recombinant vectorcontaining a polynucleotide encoding an inventive protein (which hereinis a DNA encoding an inventive protein) and the recombinant vectorcontaining the DNA encoding the GPCR;(b) a step for assaying the level of the binding of GTP to the cellmembrane fraction; and(c) a step for comparing the assayed level of this GTP binding with theassayed level of the GTP binding to the cell membrane fraction which hasnot been brought into contact with the test substance, whereby selectinga test substance capable of altering the assayed level of the GTPbinding to the cell membrane fraction.

When a cell expressing a GPCR and a G protein is stimulated by a GPCRligand, then a GTP is bound to a G protein α subunit. This phenomenon isobserved also in a membrane fraction of a cell which expresses a GPCRand a G protein. Accordingly, in the sixth method, a substance whichincreases the level of the binding of a GTP to this cell membranefraction can be selected as a GPCR agonist.

Usually, a GTP bound to a G protein α subunit is decomposed into a GDP.Accordingly, a GTP analogue which is capable of binding to an inventiveprotein but is not decomposed by a GTPase is used to measure the levelof the binding of this GTP analogue to an inventive protein, wherebyassaying the level of the binding of the GTP to the inventive protein.Such a GTP analogue may for example be a GTPγS, G_(PP)NH_(p) and thelike.

For measuring the level of the binding of a GTP analogue to a cellmembrane fraction, the GTP analogue is labeled for example with aradiolabel, and then the labeled GTP analogue is added to the cellmembrane fraction and incubated for a certain period, and then theradioactivity in the cell membrane fraction is measured by ascintillation counter and the like.

The methods for preparing and characterizing a cell membrane fractionare as described above.

A test substance which increases a level of the binding of a GTP to acell membrane fraction in a test cell which has been brought intocontact with a test substance for example by about 25%, preferably about50%, more preferably about 100%, when compared with a test cell whichhas not been brought into contact with a test substance can be selectedas a candidate GPCR agonist.

Otherwise, the aspect is similar to that in of the first methoddiscussed above.

<Seventh Method>

A method for screening for a substance capable of regulating a signaltransduction mediated by a GPCR and an inventive protein comprising:

(a) a step for bringing a test substance and a GPCR ligand into contactwitha cell membrane fraction of a cell having a recombinant vectorcontaining an inventive polynucleotide (which herein is a DNA encodingan inventive protein) and a cell membrane fraction of a cell having arecombinant vector containing a DNA encoding a GPCR, ora cell membrane fraction of a cell having the recombinant vectorcontaining a polynucleotide encoding an inventive protein (which hereinis a DNA encoding an inventive protein) and the recombinant vectorcontaining the DNA encoding the GPCR;(b) a step for assaying the level of the binding of GTP to this cellmembrane fraction; and(c) a step for comparing the assayed level of this GTP binding with theassayed level of the GTP binding in the cell membrane fraction which hasnot been brought into contact with the test substance but has beenbrought into contact with said ligand, whereby selecting a testsubstance capable of altering the assayed level of the GTP binding tothe cell membrane fraction.

In the seventh method, a substance which activates or inhibits (orsuppresses) any stage (through the time of the binding of a GTP to a Gprotein α subunit) in a cellular signal transduction can be searchedfor, including GPCR receptor agonist and antagonist.

The percentage radioactivity in a membrane fraction which has beenbrought into contact with the both of a GPCR ligand and a test substanceis calculated, on the bases of the radioactivity in the membranefraction which has been brought into contact only with the GPCR ligandbeing regarded as 100% and the radioactivity in the membrane fractionwhich has been brought into contact with none of the GPCR ligand or thetest substance as 0%. A test substance which gives a % radioactivitywhen brought into contact with the both of a GPCR ligand and the testsubstance of 75% or less, preferably 50% or less, especially 25% or lesscan be selected as a candidate of the cellular signal transductioninhibitor or suppressor. On the other hand, a test substance whichraises the percentage radioactivity when brought into contact with theboth of a GPCR ligand and the test substance to 125% or more, preferably150% or more, especially 200% or more can be selected as a candidate ofthe cellular signal transduction activator.

Otherwise, the aspect is similar to that in of the sixth methoddiscussed above. A ligand which can be employed and the ratio betweenthe ligand and a test substance are similar to those in the fourthmethod.

<Eighth Method>

A method for screening for a substance capable of regulating a signaltransduction mediated by a GPCR and an inventive protein comprising:

(a) a step for bringing a test substance into contact with a test cellcapable of expressing a protein of the invention;(b) a step for measuring the expression level of the protein of theinvention in the test cell; and(c) a step for comparing this expression level with the expression levelof said protein in the test cell which has not been brought into contactwith the test substance, whereby selecting a test substance capable ofaltering the expression level of said protein in the test cell.

In this method, a substance which activates or inhibits a signaltransduction mediated by an inventive protein by increasing or reducingthe expression of the inventive protein can be selected.

The expression level of an inventive protein can be determined bymeasuring the level of the corresponding mRNA. The level of this mRNAcan be determined by a known method such as a Northern blotting using aninventive probe described above or a PCR using inventive primersdescribed above.

Specifically, a Northern blotting can be conducted in such a manner thatan RNA is prepared from a test cell by a standard method, transferredonto a nylon membrane and the like, hybridized with a probe labeled forexample with a radioisotope or fluorescent substance, and then a doublestrand of the probe with the RNA is detected by a method suitable forthe label. A PCR can be conducted in such a manner that a cDNA isprepared from a mRNA of a test cell and used as a template to perform aPCR by a standard method using an inventive oligonucleotide set asprimers.

The expression level of an inventive protein can be determined byquantifying the protein directly. The level of this protein can bedetermined by a known method such as a Western blotting using aninventive antibody.

As a cell expressing an inventive protein, a mammalian cell, preferablya human cell is employed. It is preferred particularly to use a cellderived from a human brain, thymus, testes, spleen, small intestine,uterus and heart.

<Inventive Screening Kit>

A first inventive kit for screening for a substance capable ofregulating a signal transduction mediated by a GPCR and a proteinaccording to the invention comprises a test cell having a recombinantvector containing an inventive polynucleotide (which herein is a DNA)and a reagent for measuring the G protein effector activity.

This kit can be used in the first inventive screening method describedabove. A test cell and the reagents for measuring a G protein effectoractivity are as described above. For performing the first screeningmethod using this kit, the test cell is transduced with a recombinantvector having a DNA encoding a GPCR independently. Alternatively, thetest cell may be one having a GPCR expression vector in addition to arecombinant vector having an inventive polynucleotide.

Furthermore, the first inventive screening kit may contain a controlcell which does not have a recombinant vector having an inventivepolynucleotide (which herein is a DNA) but has a control cell having arecombinant vector having a DNA encoding a GPCR. In such a case, it canbe used in the second inventive screening method.

Moreover, the first inventive screening kit may contain a control cellwhich does not have a recombinant vector having a DNA encoding a GPCRbut has an inventive polynucleotide (which herein is a DNA). In such acase, it can be used in the third inventive screening method.

The first inventive screening kit may further contain a GPCR ligand. Insuch a case, it can be used in the fourth inventive screening method. Aligand is employed also as described above.

The first inventive screening kit may further contain a GPCR ligand anda control cell having no recombinant vector having a DNA encoding a GPCRbut having a recombinant vector having an inventive polynucleotide(which herein is a DNA). In such a case, it can be used in the fifthinventive screening method.

The second inventive kit for screening for a substance capable ofregulating a signal transduction mediated by a GPCR and a proteinaccording to the invention comprises a cell having a recombinant vectorcontaining an inventive polynucleotide (which herein is a DNA); and aGTP analogue which can bind to the protein of the invention but can notbe cleaved by a GTPase. This kit can be employed in the sixth inventivescreening method. A GTP analogue is employed also as described above.

For performing the sixth screening method using this kit, a cell istransduced with a recombinant vector having a DNA encoding a GPCRindependently. Alternatively, such a cell may be one having a GPCRexpression vector in addition to a recombinant vector having aninventive DNA.

The third inventive kit for screening for a substance capable ofregulating a signal transduction mediated by a GPCR and a proteinaccording to the invention comprises a cell having a recombinant vectorcontaining an inventive polynucleotide (which herein is a DNA) and aGPCR expression vector; and a GTP analogue which can bind to the proteinof the invention but can not be cleaved by a GTPase. This kit can beemployed in the sixth inventive screening method.

Each of the second and third screening kits according to the inventionmay further contain a GPCR ligand. In such a case, it can used in the7th inventive screening method.

The fourth inventive kit for screening for a substance capable ofregulating a signal transduction mediated by a GPCR and a proteinaccording to the invention comprises a cell capable of expressing aprotein of the invention; as well as an inventive probe, inventiveprimers or inventive antibody. This kit can be used in the 8th inventivescreening method.

<Pharmaceuticals>

A protein and an antibody according to the invention can be used aspharmaceuticals by being administered in an effective amount to a mammalsuch as a human in the forms described below.

A protein and an antibody according to the invention can be formulatedinto pharmaceutical composition in a mixture with inactive carriers,such as pharmaceutically acceptable carriers (including excipient,extender, binder, lubricant and the like) as well as customaryadditives. Such a pharmaceutical composition may be given orally orparenterally depending on the dosage form (oral formulation such astablet, pill, capsule, powder, granule, syrup and the like; parenteralformulation such as injection formulation, drip infusion formulation,dermal formulation, suppository and the like). The dose may varydepending on the type of the active ingredient, administration route,subject and the age, body weight and conditions of the patient, and maybe about 0.01 to 100 mg a day, which can be given all at once or inseveral portions.

A polynucleotide, antisense polynucleotide and ribozyme of the inventioncan be administered in an effective amount to a mammal such as a humanas pharmaceuticals in the forms of the pharmaceutical compositionsdescribed above. Otherwise, they can be introduced into a cell of asubject utilizing a liposome delivery system employing a liposome inwhich a drug to be delivered is encapsulated, a microinjection method, adirect injection method, an gene gun and the like. Also in such cases,the dose and the administration mode can be selected appropriately bythose skilled in the art, although it may vary depending on the age,body weight and conditions of the patient.

An inventive polynucleotide can be introduced into a target cell also byintegrating into a virus vector for a gene therapy.

A substance capable of regulating a signal transduction obtained by aninventive screening method can be administered in an effective amount toa mammal such as a human in the forms of the pharmaceutical compositionsdescribed above. When this substance is one encoded by a DNA, it can beintroduced into a target cell also by integrating into a virus vectorfor a gene therapy.

Effect of the Invention

According to the invention, a protein which can be regarded as a novel Gprotein involved in a cellular signal transduction and a polynucleotideencoding the same are provided.

Moreover, an inventive protein can be regarded as a protein involved ina signal transduction mediated by a GPCR and a G protein involved in thedifferentiation and the proliferation of a cell, since the G proteineffector activity in a cell having vectors expressing the GPCR and theinventive protein respectively is higher than the relevant effectoractivity in a cell having no vector expressing the inventive protein.

Furthermore, an inventive protein is considered to be one of G proteins,since it has regions having a high homology with the amino acid sequenceconserved as a GTP binding site and a GTPase activation site among Gproteins and the amino acid sequence of a trimer forming domainconserved among G proteins.

Accordingly, an inventive protein and a polynucleotide encoding the samecan preferably be employed as a regulator of an intracellular signaltransduction mediated by a GPCR and the inventive protein. Moreover, itis useful in treating or preventing a disease caused by the abnormalityin this cellular signal transduction. Specifically, it can preferably beemployed for treating or preventing a disease caused by an intracellularsignal transduction due to the defect, reduced expression level orreduced function of a protein of the invention.

Moreover, an inventive polynucleotide can preferably used in thescreening for a substance capable of regulating a signal transductionmediated by a GPCR stimulation.

Inventive antibody, antisense and ribozyme are employed preferably asregulators of an intracellular signal transduction mediated by a GPCRand an inventive protein. In addition, they can be used preferably intreating or preventing a disease caused by an abnormality in thisintracellular signal transduction. They are useful preferably for thepurpose especially of suppressing the increased abnormality in thisintracellular signal transduction.

Furthermore, an antibody of the invention can preferably be used also inthe affinity chromatography for purifying an inventive protein as wellas in the screening for a substance which may affect the expression ofan inventive protein.

A substance obtained by a screening method of the invention can be usedas a regulator of a signal transduction mediated by a GPCR and aninventive protein. In addition, it can be used preferably in treating orpreventing a disease caused by an abnormality in this intracellularsignal transduction.

An inventive oligonucleotide can preferably be used in diagnosing of adisease caused by an intracellular signal transduction due to the defectof, and abnormally increased or reduced expression level of a protein ofthe invention.

Since an inventive protein is found to be expressed in the tissue of abrain such as a cerebrum, the inventive protein, polynucleotide,antibody, antisense, ribozyme and a substance obtained by an inventivescreening method are considered to be useful in preventing or treating aneuropathy and the like. An inventive oligonucleotide is also consideredto be useful in diagnosing a neuropathy.

Also since an inventive protein is found to be expressed in a heart, theinventive protein, polynucleotide, antibody, antisense, ribozyme and asubstance obtained by an inventive screening method are considered to beuseful in preventing or treating a cardiac disease and the like(Targets, 2002, vol. 1, p 206-213).

Also since an inventive protein is found to be expressed in a thymus anda spleen, the inventive protein, polynucleotide, antibody, antisense,ribozyme and a substance obtained by an inventive screening method areconsidered to be useful in preventing or treating an immune disease andthe like.

EXAMPLES

The present invention is further described in the following Examples,which are not intended to restrict the invention.

In the following Examples, an inventive protein is sometimes abbreviatedas “Gm1”.

Example 1 Cloning of cDNA Encoding Human Gm1 Protein

A pCR-Gm1 which is a plasmid comprising a DNA encoding a full-lengthhuman Gm1 was prepared as described below.

20 ng of a plasmid DNA from a human brain-derived cDNA library (Takara)(pAP3neo) was employed as a template together with 10 μM of a forwardprimer: prGm1ATG (5′-ATGGGTCTGTGCTACAGTCTGCGG; SEQ ID NO:11) and 10 μMof a reverse primer prGNAL3′ (5′-TCACAAGAGCTCATACTGCTT; SEQ ID NO:12) aswell as TAKARA LA Taq polymerase (TAKARA LA Taq with GC Buffer, Takara)to perform a PCR to obtain an amplified DNA.

The PCR condition involved 35 cycles, each cycle involving incubationsat 95° C. for 30 seconds followed by 60° C. for 30 seconds followed by72° C. for 2 minutes.

The resultant DNA was subjected to an agarose gel electrophoresisfollowed by a purification with a QIAquick Gel Extraction kit (QIAGEN),and then recovered. This purified and recovered DNA was used as aninsert DNA.

Subsequently, a TOPO TA Cloning Kit (Invitorogen) was used and theattached protocol was followed to insert the insert DNA (50 ng) into acloning site of a pCR2.1-TOPO vector (10 ng), whereby obtaining apCR-Gm1.

The DNA thus obtained was subjected to an ABI377 DNA sequencer todetermine the nucleotide sequence, and was revealed to contain thenucleotide sequence of the nucleotide Nos. 1-1377 in the nucleotidesequence represented by SEQ ID NO:2 and to encode the full-length aminoacid sequence represented by SEQ ID NO:1.

Example 2 Detection of Expression Profile of Nucleic Acid Encoding Gm1

In order to amplify a nucleic acid encoding Gm1 specifically, a forwardprimer prGm1rt-5′ (5′-ATGGGGTGTTTGGGCGGCAACA; SEQ ID NO:13) and areverse primer prGm1rt-3′ (5′-ACGATGGTGCTTTTCCCAGACTCACCAGCCCCGAGCA; SEQID NO:14) were produced. Each 1 μg of human bone marrow-derived totalRNA (Ambion), human brain-derived total RNA (Ambion), humanspreen-derived total RNA (Ambion), human thymus-derived total RNA(Ambion), human small intestine-derived total RNA (Ambion), humanliver-derived total RNA (Ambion), human placenta-derived total RNA(Ambion), human cervix-derived total RNA (Ambion), human uterus-derivedtotal RNA (Ambion), human heart-derived total RNA (Ambion), humanskeletal muscle-derived total RNA (Ambion), human testis-derived totalRNA (Ambion) and human kidney-derived total RNA (Ambion) were employedas templates together with each 10 μM of the primer set described aboveand a SuperScript One-Step RT-PCR System (Invitrogen) and the attachedprotocol was followed to conduct a RT-PCR to amplify a mRNA. Thecondition of the RT-PCR involved an incubation at 55° C. for 30 minutesfor a reverse transcription reaction, followed by 35 cycles, each cycleinvolving incubations at 94° C. for 20 seconds followed by 60° C. for 30seconds followed by 72° C. for 1 minute.

Then, 20 μl of the RT-PCR product was subjected to an agarose gelelectrophoresis, stained with ethidium bromide, and irradiated with UVto identify the signals amplified specifically. The gel photograph isshown in FIG. 1. As evident from FIG. 1, Gm1 is expressed highly inbrain, thymus, testis, spleen, small intestine, uterus and heart.

Example 3 In Situ Hybridization in Brain Tissue (Detailed Analysis ofExpression Profile of Nucleic Acid Encoding Inventive Protein in BrainTissue)

The expression profile of a nucleic acid encoding the inventive proteinin brain tissue was investigated by the following method.

For conducting an in situ hybridization in a mouse brain tissue, thefollowing procedure was employed to clone a cDNA in the 5′ terminalregion of a mouse Gm1 gene.

20 ng of a mouse brain-derived cDNA (Clontech) was employed as atemplate together with 10 μM of a forward primer prmGm1-1(5′-ATGGGCCTATGCTACAGCCTGCGGCCGCT; SEQ ID NO:15) and 10 μM of a reverseprimer prmGm1-2 (5′-GCTGCAGGTCCCGCTTCTGCTCGCGCAGCATGCGGT; SEQ ID NO:16)as well as TAKARA LA Taq polymerase (TAKARA LA Taq with GC Buffer,Takara) to perform a PCR to obtain an amplified DNA.

The PCR condition involved 35 cycles, each cycle involving incubationsat 95° C. for 30 seconds followed by 60° C. for 30 seconds followed by72° C. for 2 minutes. The resultant DNA was subjected to an agarose gelelectrophoresis followed by a purification with a QIAquick GelExtraction kit (QIAGEN), and then recovered. This purified and recoveredDNA was used as an insert DNA.

Then, a QIAGEN PCR Cloning kit (QIAGEN) was used following to itsattached protocol to insert the insert DNA (50 ng) into a cloning siteof a pDrive vector (10 ng), whereby producing a pDrmGm1.

Similarly, a QIAGEN PCR Cloning kit (QIAGEN) was used following to itsattached protocol to insert the insert DNA (50 ng) to a cloning site ofa pDrive vector (10 ng), whereby obtaining a pDrmGolf.

(Production of Probe for In Situ Hybridization)

1 μg of a pDrmGm1 plasmid was cleaved with a restriction enzyme HindIIIor BamHI to obtain a linear plasmid pDrmGm1/HindIII and pDrmGm1/BamHI. Lμg of a pDrmGm1/HindIII, 2 μl of a DlGRNALabelingMix (Roche, Diagnostic)and 1 μl of a SP6RNA polymerase (Roche, Diagnostic) were mixed andincubated at 37° C. for 3 hours in the presence of the attached buffer.Then, 1 μl of a DNaseI (Roche, Diagnostic) was added and the mixture wasincubated at 37° C. for 30 minutes to obtain a cRNA. This cRNA wasprecipitated with ethanol, suspended in 20 μl of a TE buffer, and usedas an mGm1 sense cRNA.

Similarly, 1 μg of a pDrmGm1/BamHI, 2 μl of a DIGRNALabelingMix (Roche,Diagnostic) and 1 μl of SP6RNA polymerase (Roche, Diagnostic) weremixed, and incubated at 37° C. for 3 hours in the presence of theattached buffer. Then, 1 μl of a DnaseI (Roche, Diagnostic) was addedand incubated at 37° C. for 30 minutes to obtain a cRNA. This cRNA wasprecipitated with ethanol, suspended in a 20 μl of TE buffer, and usedas an mGm1 antisense cRNA.

(Detection by In Situ Hybridization)

A detailed analysis of the expression profile of mRNA of the Gm1 in abrain is conducted by an in situ hybridization using a labeled cRNA[Simmons et al., J. Histotechnol. 12:169-181 (1989)]. Thus, from a mousebrain fixed using paraformaldehyde and glutaraldehyde by a known method,a brain section whose thickness is 50 μm is prepared using a brainsection producing device (sliding microtome), and then adsorbed on aglass slide and dried. The brain section is made free from the paraffin,autoclaved in a target solution (Daco) (105° C., 10 minutes),dehydrated, and dried in the air. The hybridization with a probe (100 ngcRNA) is conducted in a hybridization buffer (40% formamide, 4×SSC, 1 mMEDTA, 250 μg/ml yeast tRNA, 1×Denhardt's solution, 10% dextran sulfate)at 60° C. overnight. Thereafter, the brain section is washed at 65° C.with 2×SSC, 0.1% SDS solution, treated with an RNaseA (10 μg/ml, 37° C.,30 minutes), washed with 2×SSC, 50% formamide solution, dehydrated,dried in the air, and subjected to a mRNA detection using a D1G labeledantibody detection kit (Daco).

Example 4 Construction of Expression Plasmid for Expression of Human Gm1Protein in E. coli

In order to express a large amount of a human Gm1 protein in E. coli, ahuman Gm1 protein is first expressed as a fusion protein with aglutathion S transferase, and then only the part of the human Gm1protein is cut out from the fusion protein.

Thus, the human Gm1 cDNA fragment-containing plasmid pCR-Gm1 obtained inExample 1 is double-digested with EcoRV and SpeI, and imparted with ablunt end with a Blunting Kit (Takara). The resultant DNA is subjectedto an agarose gel electrophoresis and then purified using a QIAquick GelExtraction Kit (QIAGEN) and then recovered. The recovered DNA is used asan insert DNA. The pGEX-5X-1 which had been cleaved with EcoRV and thenBAP-treated is employed as a vector, and 50 ng of this vector and 10 ngof the insert DNA are ligated using a T4 ligase, whereby producing anexpression plasmid pGEX-Gm1.

Example 5 Purification of Recombinant Human Gm1 Protein from E. coliExpressing Glutathion S Transferase—Human Gm1 Fusion Protein

The glutathion S transferase—human Gm1 fusion protein-expressing plasmidpGEX-Gm1 obtained in Example 4 is used to transform an E. coli(Escherichia coli) JM109 strain by a calcium method. The resultanttransformant is cultured in a 50 μg/ml ampicillin (Sigma)-supplementedLB medium at 37° C., and, once the O.D.₆₀₀ becomes about 0.6 reached, 1mM (final concentration) of isopropyl-β-D-thiogalactopyranoside (IPTG)is added to induce the protein expression, and incubated for further 6hours, prior to the recovery of the cells.

The cells are disrupted with ultrasonic treatment, centrifuged at 10,000g for 5 minutes to obtain a soluble fraction. The resultant solublefraction is applied onto an anti-glutathion S transferase monoclonalantibody column (Amersham Bioscience) to purify a glutathion Stransferase—human Gm1 fusion protein. Then, the purified glutathion Stransferase—human Gm1 fusion protein is treated with an active bloodcoagulation factor X (New England Biolabo) to cut a human Gm1 proteinout.

The human Gm1 protein thus cut out is subjected sequentially to a cationexchange column (S-sepharose FF; Pharmacia), hydrophobic column(Phenyl-superose; Pharmacia), hydroxyapatite column (MITSUI TOATSUCHEMICALS), cation exchange column (MONOS; Pharmacia) to purify thehuman Gm1 protein until it shows an almost single band in an SDS-PAGEanalysis with Coomassie brilliant blue staining.

Example 6 Production of Human Gm1 Protein Partial Peptide and Productionof Anti-Human Gm1 Peptide Antibody Using this Peptide

An antibody specific to a human Gm1 protein was prepared by theprocedure shown below. A peptide consisting of 14 amino acids of theamino acid Nos. 7 to 20 in the amino acid sequence represented by SEQ IDNO:1 was synthesized.

This peptide was bound to a carrier protein KML and used as animmunogen. The resultant KML fusion peptide ptGm1 was used to immunize aNew Zealand white rabbit to produce an anti-human Gm1 peptide serum. Theimmunization was repeated 5 times. From this rabbit, an antiserum wascollected, and the antiserum was purified using a protein G column(Amersham Bioscience) to isolate an antigen-specific anti-human Gm1protein antibody.

Example 7 Construction of Expression Vector for Expression of Human Gm1Protein in Animal Cell

An expression vector for transient expression of a human Gm1 protein inan animal cell is constructed. Thus, first, the human Gm1 cDNAfragment-containing pCR-Gm1 obtained in Example 1 is double-digestedwith restriction enzymes XbaI and KpnI, and the resultant DNA fragmentis introduced into a pcDNA3.1 at XbaI site and KpnI site wherebyobtaining an expression vector pcDNA-Gm1 for a transient expression ofhuman Gm1 protein in an animal cell.

Example 8 Construction of Expression Vectors for Expression of HumanDopamine Receptor Proteins in Animal Cell

Expression vectors for transient expression in an animal cell of a humandopamine D1 receptor protein and a human dopamine D2 receptor proteinrespectively were constructed by the following procedure.

In order to amplify a DNA encoding a human dopamine D1 receptor, 20 ngof a plasmid DNA from a human brain-derived cDNA library (Takara)(pAP3neo) was employed as a template together with 10 μM of a forwardprimer prDopaminD1-5′ (5′-agctcggatccATGAGGACTCTGAACACCTCTGCCA; (SEQ IDNO:17) and 10 μM of a reverse primer prDopaminD1-3′(5′-gtgcagaattcTCATCTGCGAGTTCAGGTTGGGT; SEQ ID NO:18) as well as aTAKARA LA Taq polymerase (TAKARA LA Taq with GC Buffer, Takara) toperform a PCR.

In order to amplify a DNA encoding a human dopamine D2 receptor, 20 ngof a plasmid DNA from a human brain-derived cDNA library (Takara)(pAP3neo) was used as a template together with 10 μM of a forward primerprDopaminD2-5′ (5′-agctcggatccATGGATCCACTGAATCTGTCCTGGTATGA; SEQ IDNO:19) and 10 μM of a reverse primer prDopaminD2-3′(5′-gtgcagaattcTCAGCAGTGAAGGATCTTCTGGAAGGCCTT; SEQ ID NO:20) as well asa TAKARA LA Taq polymerase (TAKARA LA Taq with GC Buffer, Takara) toperform a PCR.

The PCR condition involved 35 cycles, each cycle involving incubationsat 95° C. for 30 seconds followed by 60° C. for 30 seconds followed by72° C. for 2 minutes. The resultant DNA was subjected to an agarose gelelectrophoresis followed by a purification with a QIAquick GelExtraction kit (QIAGEN), and then recovered. This purified and recoveredDNA was used as an insert DNA.

Each insert DNA was double-digested with BamHI and EcoRI, introducedinto a pcDNA3.1(+) at BamHI and EcoRI sites, whereby obtaining animalcell expression vectors pcDNA-DlR and pcDNA-D2R.

Example 9 Construction of Baculovirus Vector Encoding Human Gm1 Protein

First, a transfer vector for overexpressing a human Gm1 protein in aninsect cell was constructed. The human Gm1 cDNA fragment-containingpCR-Gm1 obtained in Example 1 was double-digested with restrictionenzyme XbaI and SpeI, and the resultant DNA fragment was introduced intoa pAcMP2 (Pharmingen) at XbaI site to obtain a transfer vectorpAcMP-Gm1. Then 5 μg of this transfer vector and 1 μg of a BaculovirusDNA, BaculoGold DNA (Pharmingen) were cotransfected to 2×10⁶ cells ofSf21, which were cultured at 27° C. for 5 days, and then the culturesupernatant was recovered to obtain a virus solution.

Example 10 Construction of Baculovirus Vector Encoding Human Gβ Protein

A transfer vector for overexpressing a human Gβ protein in an insectcell was constructed by the procedure described below.

In order to amplify a DNA encoding human Gβ protein, 20 ng of a plasmidDNA from a human brain-derived cDNA library (Takara) (pAP3neo) wasemployed as a template together with 10 μM of a forward primer prGb1-5′(5′-ATGAGTGAGCTTGACCAGTTACGGCA; SEQ ID NO:21), 10 μM of a reverse primerprGb1-3′ (5′-TTAGTTCCAGATCTTGAGGAAGCTAT; SEQ ID NO:22) as well as aTAKARA LA Taq polymerase (TAKARA LA Taq with GC Buffer, Takara) toperform a PCR. The PCR condition involved 35 cycles, each cycleinvolving incubations at 95° C. for 30 seconds followed by 60° C. for 30seconds followed by 72° C. for 2 minutes. The resultant DNA wassubjected to an agarose gel electrophoresis followed by a purificationwith a QIAquick Gel Extraction kit (QIAGEN), and then recovered. Thispurified and recovered DNA was used as an insert DNA. Then, a TOPO TACloning Kit (Invitorogen) was used in accordance with its attachedprotocol to insert the resultant insert DNA (50 ng) into a pCR2.1-TOPOvector (10 ng) at a cloning site whereby obtaining a pCR-Gβ.

Then, the pCR-Gβ was double-digested with restriction enzyme BamHI andNotI, and the resultant DNA fragment was introduced into a pAcMP3(Pharmingen) at BamHI and NotI sites, whereby obtaining a transfervector animal cell expression vector pAcMP-Gβ.

Then, 5 μg of this transfer vector and 1 μg of a Baculovirus DNA,BaculoGold DNA (Pharmingen) were cotransfected to 2×10⁶ cells of Sf21cell, which are cultured at 27μ for 5 days, and then the culturesupernatant was recovered to obtain a virus solution.

Example 11 Construction of Baculovirus Vector Encoding Human Gγ Protein

A transfer vector for overexpressing a human Gγ protein in an animalcell was constructed by the procedure described below.

In order to amplify a DNA encoding human Gγ protein, 20 ng of a plasmidDNA from a human brain-derived cDNA library (Takara) (pAP3neo) wasemployed as a template together with 10 μM of a forward primer prGg3-5′(5′-ATGAAAGGTGAGACCCCGGTGAACA; SEQ ID NO:23), 10 μM a reverse primerprGg3-3′ (5′-TCAGAGGAGAGCACAGAAGAACTT; SEQ ID NO:24) as well as a TAKARALA Taq polymerase (TAKARA LA Taq with GC Buffer, Takara) to perform aPCR. The PCR condition involved 35 cycles, each cycle involvingincubations at 95° C. for 30 seconds followed by 60° C. for 30 secondsfollowed by 72° C. for 2 minutes. The resultant DNA was subjected to anagarose gel electrophoresis followed by a purification with a QIAquickGel Extraction kit (QIAGEN), and then recovered. This purified andrecovered DNA was used as an insert DNA. Then, a TOPO TA Cloning Kit(Invitorogen) was used in accordance with its attached protocol toinsert each resultant insert DNA (50 ng) into a pCR2.1-TOPO vector (10ng) at a cloning site whereby obtaining a pCR-Gγ.

Then, the pCR-Gγ was double-digested with restriction enzyme XbaI andPstI, and the resultant DNA fragment was introduced into a pAcMP3(Pharmingen) at XbaI and PstI sites, whereby obtaining a transfer vectorpAcMP-Gγ. Then, 5 μg of this transfer vector and 1 μg of a BaculovirusDNA, BaculoGold DNA (Pharmingen) were cotransfected to 2×10⁶ cells ofSf21 cell, which are cultured at 27° C. for 5 days, and then the culturesupernatant was recovered to obtain a virus solution.

Example 12 Construction of Baculovirus Vectors Encoding Human DopamineD1 Receptor and Human Dopamine D2 Receptor

Transfer vectors for overexpressing a human dopamine D1 receptor proteinand a human dopamine D2 receptor protein respectively in an insect cellwere constructed by the procedure described below.

The human dopamine D1 receptor expression vector pcDNA-D1R and the humandopamine D2 receptor expression vector pcDNA-D2R obtained in Example 8were each double-digested with BamHI and EcoRI, and the resultant DNAfragments are each introduced into a pAcMP3 at BamHI and EcoRI sites,whereby obtaining transfer vectors pAcMP-D1R and pAcMP-D2R.

Then, each 5 μg of the either transfer vectors and 1 μg of a BaculovirusDNA, BaculoGold DNA (Pharmingen) were cotransfected to 2×10⁶ cells ofSf21 cell, which are cultured at 27μ for 5 days, and then the culturesupernatants were recovered to obtain virus solutions.

Example 13 High Expression of Human Gm1 Protein Using Baculovirus Vectorand Purification of the Protein

A virus solution containing human Gm1 protein expression Baculovirusobtained in Example 9 was infected at MOI5 to 2×10⁷ cells of SF21 cell,which were cultured at 27° C. Five days after the infection, the cellswere recovered and suspended in an HE/PI buffer (20 mM HEPSE, 2 mM EDTAsupplemented with 1× protenase inhibitor cocktail (NACALAITESQUE). Thecell suspension was passed through a 26 G needle 15 times to disrupt thecell membrane. The suspension was then centrifuged at 4° C. and 100×gfor 5 minutes, and the supernatant obtained was centrifuged at 4° C. and20,000×g for 30 minutes, whereby recovering a human Gm1protein-containing cell membrane fraction.

Example 14 High Expression of Human Gβ Protein and Human Gγ ProteinUsing Baculovirus Vector and Purification of the Proteins

A virus solution containing human Gβ protein expression Baculovirusobtained in Example 10 and human Gγ protein expression Baculovirusobtained in Example 11 was infected at MOI5 to 2×10⁷ cells of SF21 cell,which were cultured at 27° C. Five days after the infection, the cellswere recovered and suspended in an HE/PI buffer (20 mM HEPSE, 2 mM EDTAsupplemented with 1× Protenase inhibitor cocktail (NACALAITESQUE). Thecell suspension was passed through a 26 G needle 15 times to disrupt thecell membrane. The cell suspension was then centrifuged at 4° C. and110×g for 5 minutes, and the supernatant obtained was centrifuged at 4°C. and 20,000×g for 30 minutes, whereby recovering a cell membranefraction containing the human Gβ protein and the human Gγ protein.

Example 15 High Expression of Human Dopamine D1 Receptor Protein andHuman Dopamine D2 Receptor Gγ Protein Using Baculovirus Vector andPurification of the Proteins

A virus solution containing either human dopamine D1 receptor proteinexpression Baculovirus or human dopamine D2 receptor protein expressionBaculovirus obtained in Example 12 was infected at MOI5 to 2×10⁷ cellsof SF21 cell, which were cultured at 27° C. Five days after theinfection, the cells were recovered and suspended in an HE/PI buffer (20mM HEPSE, 2 mM EDTA supplemented with 1× Protenase inhibitor cocktail(NACALAITESQUE). The cell suspensions were passed through a 26 G needle15 times to disrupt the cell membrane. This cell suspensions were thencentrifuged at 4° C. and 110×g for 5 minutes, and the supernatantsobtained were centrifuged at 4° C. and 20,000×g for 30 minutes, wherebyrecovering a cell membrane fraction containing the human dopamine D1receptor protein or the human dopamine D2 receptor protein.

Example 16 GTP Binding Assay Using Human Gm1 Protein Expressed byBaculovirus Vector

The Gm1 protein-containing membrane fraction purified in Example 13 isemployed to conduct a GTP binding assay.

The cell membrane fraction containing 2 μg of the Gm1 protein preparedin Example 13, the cell membrane fraction containing 2 μg of the Gβprotein and the Gγ protein prepared in Example 14 and the cell membranefraction containing 2 μg of the dopamine D1 receptor protein prepared inExample 15 are suspended in 55 μl of a binding buffer (59 mM Tris, 4.8mM MgCl₂, 2 mM EDTA, 100 mM NaCl, 1 μM GDP). One μM of dopamine is addedand the mixture is incubated at 30° C. for 10 minutes. Thereafter, 200pM of [35S]GTPγS is added and the mixture is incubated at 30° C. for 30minutes.

Then 1.5 ml of a washing buffer (ice-cooled 50 mM Tris, 5 mM MgCl₂, 150mM NaCl, 0.1% BSA, 0.05% CHAPS (pH7.4)) is added and the mixture isfiltered through a glass fiber filter paper GF/F. Then this filter paperis washed three times with 1 ml of Tris (pH7.4), incubated at 65° C. for30 minutes, subjected to a liquid scintillation counter to measure theradioactivity of the [35S]GTPγS which is bound to the membrane fractiondepositing on the filter paper.

Example 17 Screening for Dopamine D1 Receptor Antagonist Using Change incAMP Level as Index

2×10⁵ Cells of CHO cell were transfected with 1 μg of the dopamine D1receptor expression vector obtained in Example 8 and the Gm1 expressionvector obtained in Example 7 (pcDNA-Gm1; 3 μg) by a lipofection methodto prepare a test cell.

Then, the cells were inoculated to each well of a 96-well plate at 3×10⁴cells/well, and cultured for about 24 hours. Then, the culture mediumwas removed, and 80 μl of 1 mM IBMX-supplemented OPTI-MEN (Invitrogen)was added to the cells, which were then incubated at 37° C. for 10minutes.

Then, 10 μM of dopamine as a GPCR ligand and 10 μM of each testsubstance (butaclamol, chlorpromazine, fluphenazine, haloperidol,SCH-23390) were added and incubated at 37° C. for 30 minutes.

Then, the reaction buffer was removed, and the cAMP level was determinedusing a HitHunter ECF cyclic AMP chemiluminescent assay kit (AppliedBiosystems). As controls, a test cell which had been brought intocontact only with the GPCR ligand at the same concentration and a testcell which had been brought into contact with nothing were examined fortheir cAMP levels in the similar manner.

A substance which gave a cAMP level percentage of 85% or less uponcontact with the ligand and the test substance was selected as a signaltransduction inhibitor (antagonist), on the basis of the cAMP level withno contact being 0% and the cAMP level with the contact only with theligand being 100% (FIG. 2).

Example 18 Screening of Dopamine D1 Receptor Using Change in cAMP Levelas Index

2×10⁵ Cells of CHO cell were transfected with the dopamine D1 receptorexpression vector (1 μg) obtained in Example 8 and the Gm1 expressionvector obtained in Example 7 (pcDNA-Gm1; 3 μg) by a lipofection methodto prepare a test cell.

Then, the cells were inoculated to each well of a 96-well plate at 3×10⁴cells/well, and cultured for about 24 hours. Then, the culture mediumwas removed, and 80 μl of 1 mM IBMX-supplemented OPTI-MEN (Invitrogen)was added to the cells, which were then incubated at 37° C. for 10minutes.

Then, 10 μM of dopamine as a GPCR ligand or 10 μM of a test substance(apomorphine, CY208-248, SKF-38393, SKF-81297) was added and incubatedat 37° C. for 30 minutes.

Then, the reaction buffer was removed, and the cAMP level was determinedusing a HitHunter ECF cyclic AMP chemiluminescent assay kit (AppliedBiosystems). As a control, a test cell which had been brought intocontact with nothing were examined for their cAMP levels in the similarmanner.

A substance which gave a cAMP level percentage of 125% or more uponcontact of the test cell with the test substance was selected as asignal transduction activator (agonist), on the basis of the cAMP levelwhen the test cell has not been brought into contact with anything being100% (FIG. 3).

Example 19 Screening Using Change in cAMP Level as Index

1×10⁶ Cells of CHO cell are transfected with the dopamine D1 receptorexpression vector obtained in Example 8 (100 ng), CRE-reporter plasmid(pCRE-luc; 20 ng; Stratagene) and the Gm1 expression vector obtained inExample 7 (pcDNA-Gm1; 30 ng) by a lipofection method to prepare a testcell.

Then the cells are inoculated to each well of a 24-well plate at 5×10³cells/well, and then cultured for about 48 hours. Then, the cells arewashed with 0.2 mM buffer (3-isobutyl-methylxanthine, 0.05% BSA, 20 mMHEPES-supplemented Hunk's buffer (pH7.4); hereinafter referred to as“reaction buffer”). Then the reaction buffer is added to the cells,which are incubated at 37° C. for 30 minutes.

Then, the reaction buffer is removed, and 0.25 ml of a fresh reactionbuffer is added to the cells, and then 1 nM dopamine as a GPCR ligandand 0.1 nM to 10 nM test substance are added and the mixture isincubated at 37° C. for 30 minutes. Then the cells are dissolved in acell lysis solution (PICK-A-GENE luciferase kit, TOYO INK), and combinedwith a luminescent substrate (PICK-A-GENE luciferase kit, TOYO INK), andexamined for the fluorescent intensity using a luminometer. As controls,a test cell which had been brought into contact only with the GPCRligand at the same concentration and a test cell which had been broughtinto contact with nothing are examined for their fluorescent intensitiesin the similar manner.

A substance which gave a fluorescence intensity of percentage of 50% orless, or 150% or more upon contact with the ligand and the testsubstance is selected as a signal transduction regulating substance, onthe basis of the fluorescent intensity with no contact being 0% and thefluorescent intensity with the contact only with the ligand being 100%.

Example 20 Construction of Expression Vector for Expression of HumanAdenosine A2a Receptor Protein in Animal Cell

An expression vector for transient expression of a human adenosine A2areceptor protein in an animal cell was constructed by the proceduredescribed below.

In order to amplify a DNA encoding a human adenosine A2a receptor, 20 ngof a plasmid DNA from a human brain-derived cDNA library (Takara)(pAP3neo) was employed as a template together with 10 μM of a forwardprimer prAdenosineA2A-5′(5′-agctcggatccATGCCCATCATGGGCTCCTCGGTGTA; SEQID NO:33) and 10 μM of a reverse primerprAdenosineA2A-3′(5′-gtgcagaattcTCAGGACACTCCTGCTCCATCCT; SEQ ID NO:34)as well as a TAKARA LA Taq polymerase (TAKARA LA Taq with GC Buffer,Takara) to perform a PCR.

The PCR condition involved 35 cycles, each cycle involving incubationsat 95° C. for 30 seconds followed by 60° C. for 30 seconds followed by72° C. for 2 minutes. The resultant DNA was subjected to an agarose gelelectrophoresis followed by a purification with a QIAquick GelExtraction kit (QIAGEN), and then recovered. This purified and recoveredDNA was used as an insert DNA.

Each insert DNA was double-digested with BamHI and EcoRI, introducedinto a pcDNA3.1(+) at BamHI and EcoRI sites, whereby obtaining an animalcell expression vector pcDNA-A2a.

Example 21 Screening of Adenosine A2a Receptor Antagonist Using Changein cAMP Level as Index

2×10⁵ Cells of CHO cell were transfected with the adenosine A2a receptorexpression vector (1 μg) obtained in Example 20 and the Gm1 expressionvector obtained in Example 7 (pcDNA-Gm1; 3 μg) by a lipofection methodto prepare a test cell.

Then, the cells were inoculated to each well of a 96-well plate at 3×10⁴cells/well, and cultured for about 24 hours. Then, the culture mediumwas removed, and 80 μl of 1 mM IBMX-supplemented OPTI-MEN (Invitrogen)was added to the cells, which were then incubated at 37° C. for 10minutes.

Then, 10 μM of adenosine as a GPCR ligand and 10 μM of a test substance(DMPX) were added and incubated at 37° C. for 30 minutes.

Then, the reaction buffer was removed, and the cAMP level was determinedusing a HitHunter ECF cyclic AMP chemiluminescent assay kit (AppliedBiosystems). As controls, a test cell which had been brought intocontact only with the GPCR ligand at the same concentration and a testcell which had been brought into contact with nothing were examined fortheir cAMP levels in the similar manner.

A substance which gave a cAMP level percentage of 85% or less uponcontact of the cell with the ligand and the test substance was selectedas a signal transduction inhibitor (antagonist), on the basis of thecAMP level with no contact being 0% and the cAMP level with the contactof the cell only with the ligand being 100% (FIG. 4).

Example 22 Screening of Adenosine A2a Receptor Agonist Using Change incAMP Level as Index

2×10⁵ Cells of CHO cell were transfected with the adenosine A2a receptorexpression vector (1 μg) obtained in Example 20 and the Gm1 expressionvector obtained in Example 7 (pcDNA-Gm1; 3 μg) by a lipofection methodto prepare a test cell.

Then, the cells were inoculated to each well of a 96-well plate at 3×10⁴cells/well, and cultured for about 24 hours. Then, the culture mediumwas removed, and 80 μl of 1 mM IBMX-supplemented OPTI-MEN (Invitrogen)was added to the cells, which were then incubated at 37° C. for 10minutes.

Then, 10 μM of adenosine as a GPCR ligand or 10 μM of a test substance(CGS-21680) was added and incubated at 37° C. for 30 minutes.

Then, the reaction buffer was removed, and the cAMP level was determinedusing a HitHunter ECF cyclic AMP chemiluminescent assay kit (AppliedBiosystems). As a control, a test cell which had been brought intocontact with nothing were examined for their cAMP levels in the similarmanner.

A substance which gave a cAMP level percentage of 125% or more uponcontact with the test substance was selected as a signal transductionactivator (agonist), on the basis of the cAMP level when the test cellhas not been brought into contact with anything being 100% (FIG. 5).

Example 23 Screening by GTP Binding Assay

The cell membrane fraction containing 2 μg of the Gm1 protein preparedin Example 13, the cell membrane fraction containing 2 μg of the Gβprotein and Gγ protein prepared in Example 14 and the cell membranefraction containing 2 μg of the dopamine D1 receptor protein prepared inExample 15 are suspended in 55 μl of a binding buffer (59 mM Tris, 4.8mM MgCl₂, 2 mM EDTA, 100 mM NaCl, 1 μM GDP). 1 μM of dopamine and 0.1nM-10 nM of a test substance (for example, 1 nM SCH-23390) are added andthe mixture is incubated at 30° C. for 10 minutes. Thereafter, 200 μM of[35S]GTPγS is added and the mixture is incubated at 30° C. for 30minutes.

Then 1.5 ml of a washing buffer (ice-cooled 50 mM Tris, 5 mM MgCl₂, 150mM NaCl, 0.1% BSA, 0.05% CHAPS (pH7.4)) is added and the mixture isfiltered through a glass fiber filter paper GF/F. Then this filter paperis washed three times with 1 ml of Tris (pH7.4), incubated at 65° C. for30 minutes, subjected to a liquid scintillation counter to measure theradioactivity of the [35S]GTPγS which is bound to the membrane fractiondepositing on the filter paper.

A substance whose radioactivity percentage is calculated to be 50% orless, or 150% or more upon contact with the both of the ligand and thetest substance is selected as a substance capable of regulating thesignal transduction, on the basis of the radioactivity with the additiononly of the ligand being 100% and the radioactivity without the additionof the ligand or the test substance being 0%.

Example 24 Assay of Activation of Signal Transduction Pathway Mediatedby Gm1 Using Change in cAMP Level as Index

2×10⁵ Cells of CHO cell were transfected with the dopamine D1 receptorexpression vector (1 μg) obtained in Example 8 and the Gm1 expressionvector obtained in Example 7 (pcDNA-Gm1; 3 μg) by a lipofection methodto prepare a test cell.

Then, the cells were inoculated to each well of a 96-well plate at 3×10⁴cells/well, and cultured for about 24 hours. Then, the culture mediumwas removed, and 80 μl of 1 mM IBMX-supplemented OPTI-MEN (Invitrogen)was added to the cells, which were then incubated at 37° C. for 10minutes.

Then, 10 μM of dopamine as a GPCR ligand was added and incubated at 37°C. for 30 minutes.

Then, the reaction buffer was removed, and the cAMP level was determinedusing a HitHunter ECF cyclic AMP chemiluminescent assay kit (AppliedBiosystems). As a control, a test cell which had been brought intocontact with nothing were examined for their cAMP levels in the similarmanner.

The cAMP level of the test cell expressing the Gm1 and the dopamine D1receptor was determined on the basis of the cAMP level upon no contactbeing regarded to be 0% while the cAMP level of the control cellexpressing only the dopamine D1 receptor being regarded to be 100%, andwas revealed to be 127%. Therefore, it was proven that the Gm1-mediatedsignal transduction system do exist (FIG. 6).

Example 25 Assay of Activation of Signal Transduction Pathway Mediatedby Gm1 Using Change in cAMP Level as Index

2×10⁵ Cells of CHO cell were transfected with the adenosine A2a receptorexpression vector (1 μg) obtained in Example 8 and the Gm1 expressionvector obtained in Example 7 (pcDNA-Gm1; 3 μg) by a lipofection methodto prepare a test cell.

Then, the cells were inoculated to each well of a 96-well plate at 3×10⁴cells/well, and cultured for about 24 hours. Then, the culture mediumwas removed, and 80 μl of 1 mM IBMX-supplemented OPTI-MEN (Invitrogen)was added to the cells, which were then incubated at 37° C. for 10minutes.

Then, 10 mM of adenosine as a GPCR ligand was added and incubated at 37°C. for 30 minutes.

Then, the reaction buffer was removed, and the cAMP level was determinedusing a HitHunter ECF cyclic AMP chemiluminescent assay kit (AppliedBiosystems). As a control, a test cell which had been brought intocontact with nothing were examined for their cAMP levels in the similarmanner.

The cAMP level when allowing the Gm1 and the adenosine A2a receptor tobe expressed was determined on the basis of the cAMP level upon nocontact being regarded to be 0% while the cAMP level of the cell allowedto express only the adenosine A2a receptor being regarded to be 100%,and was revealed to be 134%. Therefore, it was proven that theGm1-mediated signal transduction system do exist (FIG. 7).

Example 26 Cloning of Mouse Gm1 Protein-Encoding cDNA

A pDr-mGm1 which is a plasmid having a DNA encoding the full-lengthmouse Gm1 was produced as described below.

20 ng of a mouse brain-derived cDNA library (Clontech) was employed as atemplate together with 10 μM of a forward primer(5′-ATGGGCCTATGCTACAGCCTGCGGCCGCT; SEQ ID NO:29) and 10 μM of a reverseprimer prmGm1STOP (5′-TCACAAGAGTTCGTACTGCTTGAGATGCATTCT; SEQ ID NO:30)as well as a TAKARA LA Taq polymerase (TAKARA LA Taq with GC Buffer,Takara) to conduct a PCR to obtain an amplified DNA.

The PCR condition involved 35 cycles, each cycle involving incubationsat 95° C. for 30 seconds followed by 60° C. for 30 seconds followed by72° C. for 2 minutes. The resultant DNA was subjected to an agarose gelelectrophoresis followed by a purification with a QIAquick GelExtraction kit (QIAGEN), and then recovered. This purified and recoveredDNA was used as an insert DNA.

Then, a QIAGEN PCR Cloning kit (QIAGEN) was used following to itsattached protocol to insert the insert DNA (50 ng) into a cloning siteof a pDrive vector (25 ng), whereby producing a pDr-mGm1.

The DNA thus obtained was subjected to an ABI377DNA sequencer todetermine the nucleotide sequence, which was revealed to contain thenucleotide Nos. 1 to 1347 in the nucleotide sequence represented by SEQID NO:27 and to encode the full-length amino acid sequence representedby SEQ ID NO:25.

Example 27 Cloning of Rat Gm1 Protein-Encoding cDNA

A pDr-rGm1 which is a plasmid having a DNA encoding the full-length ratGm1 was produced as described below.

20 ng of a rat brain-derived cDNA library (Clontech) was employed as atemplate together with 10 μM of a forward primer prrGm1ATG(5′-ATGGGCCTGTGCTACAGCCTACGGCCGCTG; SEQ ID NO:31) and 10 μM of a reverseprimer prrGm1′STOP (5′-TCACAAGAGTTCGTACTGCTTGAGGTGCATTCT; SEQ ID NO:32)as well as a TAKARA LA Taq polymerase (TAKARA LA Taq with GC Buffer,Takara) to conduct a PCR to obtain an amplified DNA.

The PCR condition involved 35 cycles, each cycle involving incubationsat 95° C. for 30 seconds followed by 60° C. for 30 seconds followed by72° C. for 2 minutes. The resultant DNA was subjected to an agarose gelelectrophoresis followed by a purification with a QIAquick GelExtraction kit (QIAGEN), and then recovered. This purified and recoveredDNA was used as an insert DNA.

Then, a QIAGEN PCR Cloning kit (QIAGEN) was used following to itsattached protocol to insert the insert DNA (50 ng) into a cloning siteof a pDrive vector (25 ng), whereby producing a pDr-rGm1.

The DNA thus obtained was subjected to an ABI377DNA sequencer todetermine the nucleotide sequence, which was revealed to contain thenucleotide Nos. 1 to 1353 in the nucleotide sequence represented by SEQID NO:28 and to encode the full-length amino acid sequence representedby SEQ ID NO:26.

Free Text in Sequence Listing SEQ ID NO:3

an example of the ribozyme of the present invention

SEQ ID NO:4

an example of the ribozyme of the present invention

SEQ ID NO:5

an example of the ribozyme of the present invention

SEQ ID NO:6

an example of the ribozyme of the present invention

SEQ ID NO:7

an example of the ribozyme of the present invention

SEQ ID NO:8

an example of the ribozyme of the present invention

SEQ ID NO:9

an example of the oligonucleotide of the present invention

SEQ ID NO:10

an example of the oligonucleotide of the present invention

SEQ ID NO:11

a primer used in an example of the present invention

SEQ ID NO:12

a primer used in an example of the present invention

SEQ ID NO:13

a primer used in an example of the present invention

SEQ ID NO:14

a primer used in an example of the present invention

SEQ ID NO:15

a primer used in an example of the present invention

SEQ ID NO:16

a primer used in an example of the present invention

SEQ ID NO:17

a primer used in an example of the present invention

SEQ ID NO:18

a primer used in an example of the present invention

SEQ ID NO:19

a primer used in an example of the present invention

SEQ ID NO:20

a primer used in an example of the present invention

SEQ ID NO:21

a primer used in an example of the present invention

SEQ ID NO:22

a primer used in an example of the present invention

SEQ ID NO:23

a primer used in an example of the present invention

SEQ ID NO:24

a primer used in an example of the present invention

SEQ ID NO:29

a primer used in an example of the present invention

SEQ ID NO:30

a primer used in an example of the present invention

SEQ ID NO:31

a primer used in an example of the present invention

SEQ ID NO:32

a primer used in an example of the present invention

SEQ ID NO:33

a primer used in an example of the present invention

SEQ ID NO:34

a primer used in an example of the present invention

1. A protein comprising any amino acid sequence selected from the groupconsisting of: (a) the amino acid sequence represented by SEQ ID NO:1;(b) an amino acid sequence of a protein involved in a G protein-coupledreceptor mediated signal transduction, the protein consisting of anamino acid sequence having a homology of 85% or more with the amino acidsequence represented by SEQ ID NO:1; (c) the amino acid sequencerepresented by SEQ ID NO:25; (d) the amino acid sequence represented bySEQ ID NO:26; (e) an amino acid sequence of a protein involved in a Gprotein-coupled receptor mediated signal transduction, the proteincomprising an amino acid sequence of amino acid Nos. 96 to 126 of SEQ IDNO:1; (f) an amino acid sequence of a protein involved in a Gprotein-coupled receptor mediated signal transduction, the proteincomprising an amino acid sequence having a homology of 95% or more withan amino acid sequence of amino acid Nos. 96 to 126 of SEQ ID NO:1; (g)an amino acid sequence of a protein involved in a G protein-coupledreceptor mediated signal transduction, the protein comprising at itsN-terminal an amino acid sequence of amino acid Nos. 1 to 126 of SEQ IDNO:1; and (h) an amino acid sequence of a protein involved in a Gprotein-coupled receptor mediated signal transduction, the proteincomprising at its N-terminal an amino acid sequence having a homology of65% or more with an amino acid sequence of amino acid Nos. 1 to 126 ofSEQ ID NO:1.
 2. A protein (1) or (2): (1) the protein consisting of theamino acid sequence represented by SEQ ID NO:1; (2) a protein involvedin a G protein-coupled receptor mediated signal transduction whichconsists of an amino acid sequence having a homology of 85% or more withan amino acid sequence represented by SEQ ID NO:1.
 3. The proteinconsisting of the amino acid sequence represented by SEQ ID NO:25. 4.The protein consisting of the amino acid sequence represented by SEQ IDNO:26.
 5. A protein (3) or (4): (3) a protein involved in a Gprotein-coupled receptor mediated signal transduction which comprises anamino acid sequence of amino acid Nos. 96 to 126 of SEQ ID NO:1; (4) aprotein involved in a G protein-coupled receptor mediated signaltransduction which comprises an amino acid sequence having a homology of95% or more with an amino acid sequence of amino acid Nos. 96 to 126 ofSEQ ID NO:1.
 6. A protein (5) or (6): (5) a protein involved in a Gprotein-coupled receptor mediated signal transduction which comprises atits N-terminal an amino acid sequence of amino acid Nos. 1 to 126 of SEQID NO:1; (6) a protein involved in a G protein-coupled receptor mediatedsignal transduction which comprises at its N-terminal an amino acidsequence having a homology of 65% or more with an amino acid sequence ofamino acid Nos. 1 to 126 of SEQ ID NO:1.
 7. A polynucleotide (9) or(10): (9) a polynucleotide encoding a protein involved in a Gprotein-coupled receptor mediated signal transduction, thepolynucleotide comprising a nucleotide sequence of nucleotide Nos. 289to 378 of SEQ ID NO:2; (10) a polynucleotide encoding a protein involvedin a G protein-coupled receptor mediated signal transduction, thepolynucleotide comprising a nucleotide sequence having a homology of 90%or more with a polynucleotide consisting of a nucleotide sequence ofnucleotide Nos. 289 to 378 of SEQ ID NO:2.
 8. A polynucleotide (11) or(12): (11) a polynucleotide encoding a protein involved in a Gprotein-coupled receptor mediated signal transduction, thepolynucleotide comprising at its 5′ terminal a nucleotide sequence ofnucleotide Nos. 1 to 378 of SEQ ID NO:2; (12) a polynucleotide encodinga protein involved in a G protein-coupled receptor mediated signaltransduction, the polynucleotide comprising at its 5′ terminal anucleotide sequence having a homology of 70% or more with apolynucleotide consisting of a nucleotide sequence of the nucleotideNos. 1 to 378 of SEQ ID NO:2.
 9. An antisense polynucleotide consistingof a polynucleotide (13) or (14): (13) a polynucleotide which inhibitsexpression of a protein according to claim 1, the polynucleotidecomprising a nucleotide sequence complementary to at least 15 contiguousnucleotides in the nucleotide sequence represented by SEQ ID NO:2; (14)a polynucleotide which inhibits the expression of a protein according toclaim 1, wherein the polynucleotide hybridizes under an intracellularcondition with a polynucleotide consisting of at least 15 contiguousnucleotides in the nucleotide sequence represented by SEQ ID NO:2.
 10. Aribozyme (15) or (16): (15) a ribozyme having an ability of cleaving apolynucleotide which comprises two polynucleotide regions complementaryto two regions respectively consisting of at least 9 contiguousnucleotides which are two regions in the nucleotide sequence representedby SEQ ID NO:2; (16) a ribozyme having an ability of cleaving apolynucleotide which comprises two polynucleotide regions whichhybridizes under an intracellular condition with two regionsrespectively consisting of at least 9 contiguous nucleotides which aretwo regions in the nucleotide sequence represented by SEQ ID NO:2; thepolynucleotide cleavable by ribosome (15) or ribosome (16) comprising anucleotide sequence selected from the group consisting of: (a) anucleotide sequence encoding the amino acid sequence represented by SEQID NO:1; (b) a nucleotide sequence encoding an amino acid sequence of aprotein involved in a G protein-coupled receptor mediated signaltransduction, wherein said protein has an ability to mediate a signaltransduction from a dopamine D1 receptor or an adenosine A2a receptor toan adenylate cyclase, and wherein said amino acid sequence has ahomology of 85% or more with the amino acid sequence of SEQ ID NO:1, andretains amino acid sequences represented by amino acid residues 119 to133, 287 to 292, 353 to 359, and 428 to 435 in the amino acid sequenceof SEQ ID NO:1; (c) a nucleotide sequence encoding the amino acidsequence represented by SEQ ID NO:25; (d) a nucleotide sequence encodingthe amino acid sequence represented by SEQ ID NO:26; (e) the nucleotidesequence represented by SEQ ID NO:2; (f) the nucleotide sequencerepresented by SEQ ID NO:27; and (g) the nucleotide sequence representedby SEQ ID NO:28.
 11. An antibody which recognizes a protein according toclaim 1 specifically.
 12. An agent for regulating a G protein-coupledreceptor mediated signal transduction containing as an active ingredienta protein according to claim
 1. 13. A therapeutic or prophylactic agentagainst a disease caused by a G protein-coupled receptor mediated signaltransduction abnormality, wherein an active ingredient of the agent is aprotein according to claim
 1. 14. An agent for regulating a Gprotein-coupled receptor mediated signal transduction containing as anactive ingredient an antisense polynucleotide according to claim
 9. 15.A therapeutic or prophylactic agent against a disease caused by a Gprotein-coupled receptor mediated signal transduction abnormality,wherein an active ingredient of the agent is an antisense polynucleotideaccording to claim
 9. 16. An agent for regulating a G protein-coupledreceptor mediated signal transduction containing as an active ingredienta ribozyme according to claim
 10. 17. A therapeutic or prophylacticagent against a disease caused by a G protein-coupled receptor mediatedsignal transduction abnormality, wherein an active ingredient of theagent is a ribozyme according to claim
 10. 18. An agent for regulating aG protein-coupled receptor mediated signal transduction containing as anactive ingredient an antibody according to claim
 11. 19. A therapeuticor prophylactic agent against a disease caused by a G protein-coupledreceptor mediated signal transduction abnormality, wherein an activeingredient of the agent is an antibody according to claim
 11. 20. Anoligonucleotide (17) or (18): (17) an oligonucleotide capable ofrecognizing a polynucleotide represented by SEQ ID NO:2 specificallywhich consists of at least 17 contiguous nucleotides in the nucleotidesequence represented by SEQ ID NO:2; (18) an oligonucleotide capable ofrecognizing a polynucleotide represented by SEQ ID NO:2 specificallywhich has a homology of 80% or more with at least 17 contiguousnucleotides in the nucleotide sequence represented by SEQ ID NO:2. 21.An oligonucleotide according to claim 20 which is used as a probe or aprimer.
 22. A method for screening for a substance capable of regulatinga signal transduction mediated by a G protein-coupled receptor and a Gprotein comprising: (a) bringing a test substance into contact with atest cell capable of expressing a protein according to claim 1; (b)measuring the expression level of the protein according to claim 1 inthe test cell; and (c) comparing the expression level with theexpression level of the protein in the test cell which has not beenbrought into contact with the test substance, to thereby select a testsubstance capable of altering the expression level of the protein in thetest cell.
 23. A kit for screening for a substance capable of regulatinga signal transduction mediated by a G protein-coupled receptor and aprotein according to claim 1, which comprises a test cell having arecombinant vector containing a polynucleotide encoding a proteinaccording to claim 1 and a reagent for measuring the G protein effectoractivity or an index value correlating therewith.
 24. A screening kitaccording to claim 23, wherein the test cell further has a recombinantvector having a DNA encoding a G protein-coupled receptor.
 25. Ascreening kit according to claim 23 further containing a Gprotein-coupled receptor ligand.
 26. A screening kit according to claim23 further containing a control cell having a recombinant vector havinga DNA encoding a G protein-coupled receptor.
 27. A kit for screening fora substance capable of regulating a signal transduction mediated by a Gprotein-coupled receptor and a protein according to claim 1, whichcomprises a test cell having a recombinant vector containing apolynucleotide encoding a protein according to claim 1 and a reagent formeasuring the G protein effector activity or an index value correlatingtherewith, the kit further containing a control cell having arecombinant vector containing a polynucleotide encoding a proteinaccording to claim
 1. 28. A kit for screening for a substance capable ofregulating a signal transduction mediated by a G protein-coupledreceptor and a protein according to claim 1, which comprises: a cellhaving a recombinant vector containing a polynucleotide encoding aprotein according to claim 1; and, a GTP analogue which can bind to theprotein according to claim 1 but cannot be cleaved by a GTPase.
 29. Ascreening kit according to claim 28 wherein the cell further has arecombinant vector having a DNA encoding a G protein-coupled receptor.30. A kit for screening for a substance capable of regulating a signaltransduction mediated by a G protein-coupled receptor and a proteinaccording to claim 1, which comprises: a cell having a recombinantvector containing a polynucleotide encoding a protein according to claim1; a cell having a recombinant vector having a DNA encoding the Gprotein-coupled receptor; and, a GTP analogue which can bind to theprotein according to claim 1 but cannot be cleaved by a GTPase.
 31. Ascreening kit according to claim 30 further containing a Gprotein-coupled receptor ligand.
 32. A kit for screening for a substancecapable of regulating a signal transduction mediated by a Gprotein-coupled receptor and a protein that comprises: a cell capable ofexpressing a protein comprising an amino acid sequence selected from SEQID NOs: 1, 25, and 26; and an agent selected from an oligonucleotidehaving at least seventeen contiguous nucleotides of SEQ ID NO: 2, and anantibody that specifically binds to the protein.